genome visualization by classic methods in light microscopy

GENOMEVISUALIZATIONby CLASSIC METHODSin LIGHTMICROSCOPY

Methods in VisualizationSeries Editor: Gérard Morel

In Situ Hybridization in Light MicroscopyGérard Morel and Annie Cavalier

Visualization of Receptors: In Situ Applicationsof Radioligand Binding

Emmanuel Moyse and Slavica M. Krantic

Genome Visualization by Classic Methods in Light MicroscopyJean-Marie Exbrayat

Imaging of Nucleic Acids and Quantificationin Phototonic Microscopy

Xavier Ronot and Yves Usson

In Situ Hybridization in Electron MicroscopyGérard Morel, Annie Cavalier,

and Lynda Williams

GENOMEVISUALIZATIONby CLASSIC METHODSin LIGHTMICROSCOPYJean-Marie Exbrayat, Ph.D., D.Sc.

Boca Raton London New York Washington, D.C.CRC Press

This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted withpermission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publishreliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materialsor for the consequences of their use.

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No claim to original U.S. Government worksInternational Standard Book Number 0-8493-0043-6

Library of Congress Card Number 00-057198Printed in the United States of America 1 2 3 4 5 6 7 8 9 0

Printed on acid-free paper

Library of Congress Cataloging-in-Publication Data

Exbrayat, J. M.Genome visualization by classic methods in light microscopy / Jean-Marie Exbrayat

p. cm. — (Methods in visualization)Includes bibliographical references and index.ISBN 0-8493-0043-6 (alk. paper)1. Nucleic acids. 2. Histochemistry. 3. Microscopy. I. Title. II. Series.

QP620 .E93 2000572.8—dc21 00-057198 CIP

0043fr_m.fm Page IV Tuesday, October 10, 2000 2:18 PM

V

Visualizing molecules inside organisms, tissues, or cells continues to be an exciting challenge forcell biologists. With new discoveries in physics, chemistry, immunology, pharmacology, molecularbiology, analytical methods, etc., limits and possibilities are expanded, not only for older visualizingmethods (photonic and electronic microscopy), but also for more recent methods (confocal andscanning tunneling microscopy). These visualization techniques have gained so much in specificityand sensitivity that many researchers are considering expansion from in-tube to

in situ

experiments.The application potentials are expanding not only in pathology applications but also in morerestricted applications such as tridimensional structural analysis or functional genomics.

This series addresses the need for information in this field by presenting theoretical andtechnical information on a wide variety of related subjects:

in situ

techniques, visualization ofstructures, localization and interaction of molecules, functional dynamism

in vitro

or

in vivo.

The tasks involved in developing these methods often deter researchers and students fromusing them. To overcome this, the techniques are presented with supporting materials such asgoverning principles, sample preparation, data analysis, and carefully selected protocols. Addi-tionally, at every step we insert guidelines, comments, and pointers on ways to increase sensitivityand specificity, as well as to reduce background noise. Consistent throughout this series is anoriginal two-column presentation with conceptual schematics, synthesizing tables, and useful com-ments that help the user to quickly locate protocols and identify limits of specific protocols withinthe parameter being investigated.

The titles in this series are written by experts who provide to both newcomers and seasonedresearchers a theoretical and practical approach to cellular biology and empower them with toolsto develop or optimize protocols and to visualize their results. The series is useful to the experiencedhistologist as well as to the student confronting identification or analytical expression problems. Itprovides technical clues that could only be available through long-time research experience.

Gérard Morel, Ph.D.

Series Editor

SERIES PREFACE

0043fr_m.fm Page V Tuesday, October 10, 2000 2:18 PM

0043fr_m.fm Page VI Tuesday, October 10, 2000 2:18 PM

VII

GENERAL INTRODUCTION

Visualization of nucleic acids has becomeindispensable to studying cells, tissues, andorganisms, to understanding development, dif-ferentiation, and physiology, and to studyingpathological disorders. Visualization is alsoused to determine the effects of a pharmaceu-tical or a toxic molecule on cells, and is oftenessential for routine examination in clinical ser-vices. Appreciation of the expression of genesin a cell is also of interest in biotechnology.

To visualize nucleic acids, most tech-niques use dyes of natural or synthetic origin.Although, the action of the dyes is not alwaysprecisely known, their affinity and specificityare no longer in doubt. More precise methodsare based upon true chemical reactions

in situ

in which a nucleic acid is one reagent and theother is a dye molecule. In these histochemicalmethods, physicochemical conditions areknown. The results of these reactions mustalways be validated by means of a negativecontrol reaction in which DNA or RNA isdeleted by use of chemical or enzymatichydrolysis.

Today, researchers have access toincreasingly more precise and more selectivemethods for visualization. Immunocytochemi-cal methods now use an antibody–antigen reac-tion, where a nucleic acid is the antigenicmolecule. With

in situ

hybridization, genes,DNA, and RNA are precisely visualized withnucleic probes. At a time in which precision isthe rule, precise quantification, aided by pow-erful computers, is always indispensable intracking gene expression. Numerous methodspermit such analysis by flux cytometry, as wellas by automatic quantitative image analysis.In this age of genetics, nucleic acid visualiza-tion is a necessary part of many scientificinvestigations.

The purpose of this book is to provideinsight into several classic techniques, histolog-ical as well as histochemical, that can be used toappreciate the nucleic acid status of the cell aswell as to provide an overview of RNA andDNA distribution in cells and tissues, more orless precisely according to the information.Some of the techniques are relatively easy toperform in a nonspecialized laboratory. Othersare more difficult. Certain techniques permitvisualization and quantification of DNA and/orRNA distribution in tissues. Several of thempermit choice of a more precise method to fur-ther explicate the genome.

Numerous methods for DNA and RNA visu-alization exist, but few recent analytical bookson the topic are available. This book presents ananalytical approach. This approach seems valu-able based on the author’s participation at thesymposia organized by Dr. Gérard Morel, theeditor of the series of which this book is a part,based on the author’s more than 20 years ofteaching in a school of technicians specializingin histology, and on the daily use of histologyfor the author’s own research.

The book is organized in eight chapters. Inthe first, visualization principles are described.The second chapter concerns preparation of tis-sues; and the third covers staining by classicdyes. In the fourth chapter, histochemical meth-ods are described. The fifth chapter concernsfluorescent dyes. In the sixth chapter, observa-tion phases are described, along with sectionmounting and a presentation on the light micro-scope. Preparation of products is given in Chap-ter 7. In the eighth chapter, some protocols thatare used in the author ’s laboratory aredescribed. The book ends with a few examplesof staining and a glossary.

0043fr_m.fm Page VII Tuesday, October 10, 2000 2:18 PM

VIII

The author thanks Dr. Gérard Morel, the series editor of Methods in Visualization, who gavenumerous suggestions for improving and expanding the manuscript. He also thanks GenevieveEscudie, Jeanne Estabel, and Paulette Pujol, who have taught histology and cytology with him forseveral years, and Marie-Therese Laurent, a skilled technician whose ability and effectivenesscontinue to contribute significantly to the improvement of methods that are used in the author’slaboratory.

ACKNOWLEDGMENTS

0043fr_m.fm Page VIII Tuesday, October 10, 2000 2:18 PM

IX

Jean-Marie Exbrayat, Ph.D., D.Sc.,

is Professor at the Catholic University in Lyon (France) wherehe manages the Laboratory of General Biology and the School of Histology. He is also Directeurd'Etudes (Professor) at the Ecole Pratique des Hautes Etudes, where he manages the Laboratoryof Vertebrate Reproduction and Development.

Professor Exbrayat obtained his M.S and Ph.D. in 1974 and 1977, respectively, from theMontpellier University (France). He was appointed assistant, then master assistant, of biology atthe Catholic University in Lyon in 1978, and became doctor of sciences in 1986. He became aprofessor in 1987 and also Directeur d'Etudes (Professor) at the Ecole Pratique des Hautes Etudes(Practical School of High Studies) in 1991.

He is a member of the New York Academy of Sciences, American Association for theAdvancement of Sciences, Societas Europaea Herpetologica, Zoological Society of France, Her-petological Society of France (of which he was General Secretary from 1991 until 1997), and theFrench Association of Histotechnology.

Professor Exbrayat is the author of more than 150 papers and four books. His current majorinterests include the variations of genital tracts and endocrine organs during reproductive cycles,as well as embryonic development in lower vertebrates.

THE AUTHOR

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0043fr_m.fm Page X Tuesday, October 10, 2000 2:18 PM

XI

CONTENTS

General Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VII

Abbreviations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

XIII

Chapter 1- Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

Chapter 2 - Tissue Preparation . . . . . . . . . . . . . . . . . . . . .

21

Chapter 3 - Staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

55

Chapter 4 - Histochemical Methods . . . . . . . . . . . . . . . . .

81

Chapter 5 - Fluorescent Methods . . . . . . . . . . . . . . . . . . .

113

Chapter 6 - Observation Phases. . . . . . . . . . . . . . . . . . . . .

127

Chapter 7 - Preparation of Products . . . . . . . . . . . . . . . . .

137

Chapter 8 - Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

155

Examples of Staining Methods . . . . . . . . . . . . . . . . . . . . .

169

Glossary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

181

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

187

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0043fr_m.fm Page XII Tuesday, October 10, 2000 2:18 PM

XIII

ABBREVIATIONS

DAPI

➫

4

′

, 6 diaminido-2-phenylindol

DDSA

➫

dodecenyl succinic anhydride

DIPI

➫

4

′

, 6-diaminido-2 imidazolinyl-

4

H-

5

H

DMP30

➫

2-4-6-tridimethylaminoethyl phenol

DNA

➫

deoxyribonucleic acid

MMA

➫

methyl methacrylate

MNA

➫

methyl nadic anhydride

mRNA

➫

messenger ribonucleic acid

PAS

➫

periodic acid and Schiff

PBS

➫

phosphate buffered salt

RNA

➫

ribonucleic acid

snRNA

➫

small nuclear ribonucleic acid

tRNA

➫

transfer ribonucleic acid

0043fr_m.fm Page XIII Tuesday, October 10, 2000 2:18 PM

0043fr_m.fm Page XIV Tuesday, October 10, 2000 2:18 PM

Chapter 1

Principles

0043C01 Page 1 Thursday, October 5, 2000 10:35 AM

Contents

3

Contents

1.1 Nucleic Acids and Histones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.1.1 DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.1.1.1 Structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.1.1.2 Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.1.1.3 Duplication of Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . .1.1.1.4 DNA in Mitochondria and Plastids . . . . . . . . . . . . . . . . . . . . . .1.1.1.5 DNA in Bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.1.1.6 Viral DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.1.2 RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.1.3 Viral Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.1.4 Nucleoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.1.5 Visualization of Nucleic Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.2 General Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.2.1 Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.2.1.1 Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.2.1.2 Phenomenon. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.2.1.3 Mechanism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.2.1.4 Acidic and Basic Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.2.1.5 Mordancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.2.2 Methods of Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.2.2.1 Staining Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.2.2.2 Basophilic Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.2.2.3 Metachromasy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.2.2.4 Visualization of Reductive Groups . . . . . . . . . . . . . . . . . . . . . .1.2.2.5 Visualization of Carbonyl Groups . . . . . . . . . . . . . . . . . . . . . . .1.2.2.6 Quantitative Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1.2.2.7 Vessels for Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

555789999

101111111111111112131313141517181919


1.1 Nucleic Acids and Histones

5


1.1.1 DNA

1.1.1.1 Structure

DNA is characterized by a sugar, deoxyribose,more particularly the 2–

D

–deoxyribofuranose.This molecule is associated with a nitrogenousbase and a molecule of phosphoric acid. Groupsbelonging to deoxyribose–phosphate are boundby phosphodiester bonds between the 3

'

and 5

'

carbons belonging to two successive deoxyri-boses (Figure 1.1).

All the bases belong to the purine or pyrimidinefamily. Puric bases are adenine and guanine;pyrimidic bases are cytosine and thymine (Figure1.2). Other bases can occur in DNA composi-tion, but they are very rarely found and theirquantities are very small. Certain molecular sitesin the bases can incur hydrogen binding and canallow binding with proteins.

adenine thymine

guanine cytosine

➫

A deoxyribose and a nitrogenous base con-stitute a deoxynucleoside (or a nucleoside).

➫

Deoxyribose, nitrogenous base, and phos-phate constitute a deoxyribonucleotide (anucleotide).

➫

By convention, carbons belonging to pen-toses are numbered from 1

'

to 3

'

. Atomsbelonging to bases are numbered from 1 to 5or from 1 to 9.

Figure 1.1 Deoxyribose.

➫

Thymine is the 5-methyl uracile.

Figure 1.2 Puric and pyrimidic bases.

HOCH2

H H HH

HOH

OHOC

C C

C1

23

4

5

NH2

NN

NNH

CHHC

C C

C

O

NH

CCHN CH3

CH O—C—

——

—

—

—

H

HNCH

H2N N

N

N

O

C

CC

C

H

CH

CH

NH2

N

NO C

C


Principles

6

The molecular structure of DNA is that of adouble helix, which was discovered by Watsonand Crick in 1953. This double helix is com-posed of two molecular chains that are linkedby nitrogenous bases. The structure correspondsto a stack of deoxynucleotides linked to eachother by diester bridges between the 3

'

and 5

'

carbons belonging to two adjacent deoxyriboses.These deoxyribose–phosphate bonds are ori-ented in such a way that gives each a helicalaspect. These stacks form the primary structureof DNA (Figure 1.3).

The two molecular chains are oriented in theopposite direction along the helix axis: one from5

'

to 3

'

, the other from 3

'

to 5

'

. These trends area dyad, and they are antiparallel.

The two halves of the DNA double helix arebound by the pairing of two complementarynitrogenous bases. Binding the two bases arehydrogen bonds which allow the double helix toopen at the moment of DNA replication or whensequences of RNA are copied.

➫

X-ray diffraction analysis provides preciseinformation about the size of the molecule: twoneighboring bases are separated by 0.34 nm;each helix turn is 3.4 nm; 10 base pairs arefound on each turn; the helix diameter is 2 nm.

➫

The entire molecule is a ladder-like structurewith bases as rungs.

A = AdenineT = ThymineG = GuanineC = Cytosine

Figure 1.3 DNA double helix. (From Smith, C.A. and Wood, E.J.,

Les Biomolecules

, Masson, 197. With permission.)

A

G C

T

G

A

C

T

T

C

C

T

CG

5' 3'

3'5'

A

G

G

A

G

C

T

A



7

Nitrogenous bases are complementary (Figure1.4). A puric base is always linked to a pyrimidicbase. DNA geometry possesses both a large anda small groove.

The DNA molecule is very stable. This stabilityis a consequence of hydrogen bonds, which indi-vidually are not very stable. However, when theyare numerous, they possess hydrophobic inter-actions between two neighboring bases and theystabilize nucleoplasmic phosphoric groups.Mg

2+

ions also contribute to the stability of theDNA molecule. In addition, the histones, basicproteins that are characteristic of eukaryoticorganisms, are bound to DNA to form nucle-oproteins, which also contribute greatly to thestability of DNA.

thymine adenine

cytosine guanine

➫

The structure described is that of

β

-DNA,which is the primary DNA

in vivo

. Othermolecular structures can be found. A-DNA isdense and possesses 11 pairs of bases per helixturn. Z-DNA has 12 pairs of bases per turn,and its general form is a zigzag.

➫

Adenine is always bound to thymine by twohydrogen bonds, and cytosine to guanine bythree.

Figure 1.4 Complementary nitrogenous bases. (From Smith, C.A. and Wood, E.J.,

Les Biomolecules

, Masson, 199. With per-mission.)

1.1.1.2 Genome

The general primary structure of DNA is thesame for all species, but the sequence of basesis different from one to another. This sequenceconstitutes the genome, i.e., the totality of genes.

➫

Nucleotide chaining is the primary DNAstructure.

HH H

H

C

C

O

O

C

C

C C

C

C

C

CN

N

N

N

N

N

N

H

H

H

H

H

H

H

H

H

H

N

N

N

N

N

H

C

C

C

C

C

O

O

C

C

C

CH

N

N

Guanine


Principles

8

Nuclear DNA never leaves the nucleus. Geneticinformation must be copied in RNA during tran-scription. Then, RNA migrates to the cytoplasmwhere it originates protein synthesis during thetranslation phase.

1.1.1.3 Duplication of chromosomes

During the S phase of the cell cycle, the doublehelix is split by the separation of paired nitrog-enous bases. Complementary nucleotides arepaired to free nucleotides contained in thenucleoplasm. A double helix resembling the ini-tial double helix is reconstituted from each DNAstrand. DNA is now replicated.

At the end of the cell cycle, at mitosis, the entirecell is involved in important alterations, charac-terized by nuclear membrane disappearance,constitution of a microtubular achromatic spin-dle, and individualization of chromosomes.Chromosomes always possess one DNA doublehelix that is linked to histones.

After individualization, the homologous chro-mosomes descending from the same DNAsequence are disposed on an equatorial plane atmetaphase. They migrate to each side of the cellfrom the center to access the two opposite poles.The center of the cell divides. The two chromo-some groups are surrounded by a new nuclearmembrane and become the nuclei of the two newcells.

➫

The cell cycle is divided into four phases asa function of DNA quantity: the G1 phase, inwhich DNA quantity is Q; the S phase (syn-thesis), during which DNA is duplicated; theG2 phase, during which DNA quantity is 2Q;and M (mitosis), during which the DNA of acell (2Q) is distributed into two new cells (1Qto each new cell).

➫

Mitosis is a complex phenomenon that can bedivided into four steps: prophase, metaphase,anaphase, and telophase (Figure 1.5).

➫

Prophase,

during which chromosomes areindividualized into the cell. At this stage, thenuclear membrane disintegrates and microtu-bules are disposed on a spindle that is said tobe achromatic because it is not stained by dyes.

➫

Metaphase,

during which the chromo-somes, always sustained by fibers of the spin-dle, are disposed on an equatorial plane, whichis a characteristic cytological picture.

prophase

metaphase



9

➫

Anaphase,

during which the chromosomesmigrate to each cell pole.

➫

Telophase,

during which the two daughtercells are separated and nuclei are individual-ized.

➫

Before dividing again, the cell is said to bein interphase. Chromatin is scattered into thenucleus.

Figure. 1.5 Mitosis.

1.1.1.4 DNA in mitochondria and plastids

These organelles contain small DNA molecules.This DNA, which is scattered in the matrix ofmitochondria and the stroma of plastids, resem-bles bacterial DNA. It is not associated withhistones. In mitochondria, DNA codes for cer-tain proteins that are characteristic of theseorganelles; the other proteins are encoded bynuclear DNA.

➫

The DNA of mitochondria and plastids can-not be detected by the classic techniques ofhistology or histochemistry.

1.1.1.5 DNA in bacteria

Bacteria possess double-stranded circular DNA.All the prokaryotes have one chromosome andoften plasmids, which are also very small circu-lar DNA.

➫

It is sometimes useful to visualize nucleicacids in prokaryote cells, especially those ofmycoplasma when infecting cell cultures.

1.1.1.6 Viral DNA

➫

See Section 1.1.3.

1.1.2 RNA

RNA is characterized by a ribose (Figure 1.6).As with DNA, this sugar is associated with onenitrogenous base and a phosphoric group.

➫

More precisely, the ribose is 2-

D

-ribofura-nose.

➫

The molecule composed of ribose and onenitrogenous base is called a ribonucleoside (ornucleoside). Binding with the phosphoricgroup is one ribonucleotide (nucleotide).

anaphase

telophase


Principles

10

RNA molecules are generally single stranded.They are generated from one single DNA strand.However, free RNA bases can be paired if theirstructure is suitable. During copying of DNA inRNA, a DNA–RNA duplex is formed after com-plementary bases have been paired.

At present, four RNA types are known. Messen-ger RNAs (mRNAs), which are copied fromDNA, bring genetic information into the cyto-plasm. This mRNA is then translated in apolypeptide into ribosomes, which are verysmall particles linked to one ribosomal RNA(rRNA). During the translation, mRNA–ribo-some complexes are paired with amino acidsthat are brought into the cytoplasm by transferRNAs (tRNAs). A fourth RNA class—the“small nuclear RNAs” (snRNAs)—is combinedwith proteins to form ribonucleoprotein parti-cles, which are required in nuclear metabolismof RNAs.

Figure 1.6 Ribose.

➫

Adenine is linked to uracil, and guanine tocytosine.

Figure 1.7 Uracil.

➫

The four RNA types are independently tran-scribed from DNA.

1.1.3 Viral Nucleic Acids

Viral genomes are very diversified. They cancomprise single or double DNA strands, whichcan be linear or circular. Viral genomes can alsobe composed of single or double RNA strands.

➫

The viral genome cannot be visualized byphotonic microscopy.

HOCH2

HH

HH

OH OH

OHO

C

C

C

C1

23

4

5

O

O C

C

CH

CHHN

NH


1.2 General Principles

11

1.1.4 Nucleoproteins

Nucleoproteins are substances arising from pro-teins and nucleic acid combinations. Their liai-sons are formed from basic groups to lateralchains linked to negative loads of phosphoricgroups belonging to nucleotides. In the nucleus,histones are associated with DNA. Nucleoli aredeprived of histones. Other deoxyribonucleictypes can be observed in the cell nucleus. Ribo-nucleoproteins arise from ribosomes.

➫

Histones possess numerous basic aminoacids, especially arginine and lysine. They donot possess tryptophan.

1.1.5 Visualization of Nucleic Acids

Visualization of nucleic acids on sections is pos-sible using visualization methods for sugar,nitrogenous bases, phosphoric groups, or evenassociated proteins. Methods for general stain-ing are useful to visualize cell nuclei: use ofspecific dyes in association with other dyes thatstain other tissues or cell elements.

1.2 GENERAL PRINCIPLES

1.2.1 Dyes

1.2.1.1 Definition

A dye is a chemical substance that is able topermanently stain a cellular or tissue compo-nent.

1.2.1.2 Phenomenon

Staining is a very complex phenomenon. It isaffected by chemical factors such as acido-basicliaisons. It is also affected by physical factorssuch as dye diffusion, capillarity, and so on.

➫

In complex staining, competition betweendyes and blinding of one dye by another canalso be observed. The phenomenon is notalways precisely known.

1.2.1.3 Mechanism

In histological staining, the result is known but is


Principles

12

obtained from empirical methods because theunderlying mechanisms have not been eluci-dated. Conversely, in histochemical methods,the effects and molecular mechanisms regardingthe action of the dye on the tissue are known,along with an understanding of the parameters,such as temperature or pH, that are necessary.

A dye is a molecule that empirically possessestwo particular chemical groups: the chro-mophore, which gives the color, and anotheratomic group, the auxochrome, which isrequired to fix the dye molecules on the tissue-specific molecules (Figure 1.8).

Most dyes are organic synthetic products; a fewhave a natural origin, essentially vegetal.

The main chromophore groups are:— Azoic–N=N–— Azine— Indamine or thiazine— Nitro— Kinonic form of aromatic molecules and naph-thokinones

The higher the number of chromophoric groups,the more intense the coloration will be.

➫

Certain substances are colored because theypossess a chromophore group. However, theydo not possess an auxochrome, and they cannotfix themselves on the tissues. These substancesare not dyes, but chromogenes.

➫

Certain chemicals are not chemically fixedon tissues, but they are dissolved into them.these substances are not dyes but lysochromes.They are used to stain lipids.

1

= Chromophore

2 = Neutral molecular part

➫

1 + 2 = Chromogene

3 = Auxochrome Mordant

Figure 1.8 Structure of dyes.

1.2.1.4 Acidic and basic dyes

In classic histology, it is recognized that acidic dyesare cytoplasmic and that basic dyes are nuclear.However, this acid/base terminology is not linkedto the pH of the dye solution, it is independent ofthe acidic or basic nature of this solution.

Tissue

Tissue

12

1

1

2

2

3

4

Tissue



13

The distinction refers to the auxochrome, whichis the part of the dye molecule that is requiredfor tissue fixation.Basic dyes possess an –NH

+3

cationic auxo-chrome. In contrast, acidic dyes possess ananionic auxochrome, such as –COO

–

or evenSO

3–

.

➫

Eosin sodium salts, which are acidic dyes,generally have a basic pH.

➫

When an SO

–3

group is fixed on a basic dye,the latter is transformed to an acidic dye (forexample, acidic and basic fuchsins).

1.2.1.5 Mordancy

When a staining substance does not have anauxochromic group, a mordant is useful. A mor-dant is a molecule that allows the fixation of thestaining substance on the tissue. Two methodscan be used. The tissue can first be submitted tothe action of this molecule and then to the stain-ing substance. Or, the mordant can be added tothe staining solution. Iron and ammonium alumare often used as mordants. Such a solution iscalled a lac.

➫

Staining with mordancy is used especially tovisualize cell nuclei with hematoxylin ornuclear fast red.

1.2.2 Methods of Staining

1.2.2.1 Staining types

Visualization of nucleic acids on sections,smears, or cell cultures can be accomplished byhistochemical reactions or basic dyes. Tissuepreparations are subjected to several operationsbefore being stained. Sections coming from waxblocks must be dewaxed by use of a solvent,then hydrated, because dyes are generally pre-pared as aqueous solutions. Sometimes, to keeptissue slices from becoming dislodged, collo-dion is used.

➫

Collodion (or celloidin) is a molecule ofnitrocellulose. It is placed on the slices beforedehydration and allows the sections to besecured in place. Because it is permeable todyes, it also permits staining. Like all nitrocel-lulose molecules, it must be stored at 4˚C andshocks must be avoided.

Nitrocellulose mole-cules can explode

.


Principles

14

Histochemical methods of visualization requirecontrolled conditions. For classic staining, nospecial precautions are required.

Staining can be performed in the progressivemode. In this case, contact between the dye andthe tissue is stopped when the staining appearsto be “good.” Alternatively, staining can be per-formed in the regressive mode. In this case, thepreparation is overstained; then, a differentiationsubstance is used that excludes the excess dye,under microscope control.

➫

Progressive modes are used for Groat hema-toxylin staining of nuclei. The regressive modeis used with numerous other hematoxylinstaining techniques, notably with Massontrichroma.

1.2.2.2 Basophilic staining

1.2.2.2.1

P

RINCIPLE

Basophilic components of tissues, such as nucleicacids, react with basic dyes. Basophilic stainingis in the category of histochemical methods inwhich the reaction between tissue or cell mole-cule and the dye is known and controlled.

1.2.2.2.2

D

EFINITION

A basic dye is a salt whose cation is colored andwhose anion is not.

1.2.2.2.3

M

ECHANISM

Basophilic staining is characterized by the fixa-tion of a colored cation on the tissue or cellelement, which is colored by the cation.

1.2.2.2.4

F

ACTORS

ACTING ON BASOPHILIC

REACTIONS

Basophilic reactions are subject to external fac-tors that must be considered during the interpre-tation phase. Competition between the dye cationand other cations can exist. These latter cationscan be uncolored or colored and they can belongto the staining solution, to the tissue, or to both.On the other hand, the staining reaction can beattenuated or suppressed if the penetration of stainmolecules into the tissue is insufficient. It isalways possible that stain molecules have fixedto other tissue groups by different binding mech-anisms (adsorption, hydrogen binding, etc.).

To be viable, a basophilic reaction must followcertain rules:� Cell and tissue acidic groups must be ionized. � Tissue and cell anions and cations must not be

➫Acidophilic staining occurs when tissuesreact with an acidic dye. The principles are thesame as in basophilic staining. These reactionsare never used to visualize nucleic acids.

➫Certain heavy dye molecules cannot accesstissue sites to stain them.



15

bound to one another.� Tissue anionic groups must be in sufficient quan-tity to be visualized.Numerous tissues and cell substances haveacidic groups: nucleic acids, proteins, sugars,and, lipids. Basophilic reactions must be con-trolled to be certain of the nature of the stainedelements.Not all basic dyes react in the same manner onall basophilic structures. Certain dyes are highlyspecific for nucleic acids.

1.2.2.2.5 PRECAUTIONS

For staining with a basic dye to succeed, someprecautions must be taken:� The dye must be as pure as possible and must notbe in a mixture.� The staining method must be progressive.Treatments after the staining are also important.� It is advisable to wash as few times as possiblewith a buffered solution at the pH of the stain. � Dehydration with 2-methyl-butan-2-ol is pre-ferred to dehydration with ethanol.This type of staining is very useful for visualiz-ing nucleic acids and proteins.

➫Methyl green and pyronin are basic dyes thatare highly specific for nucleic acids. Pyroninis used to visualize RNA, and methyl greenreacts on DNA. Both are used for methylgreen–pyronin staining.➫Certain basic dyes, like toluidine blue,jointly visualize nucleic acids and other sub-stances (see Section 1.2.2.3).

1.2.2.3 Metachromasy

1.2.2.3.1 PHENOMENON

Metachromasy is a physical quality of stainingsolutions. It permits visualization of nucleicacids, i.e., by toluidine blue, azure blue, or thio-nin. It also permits one to differentiate, by thedifferent colors, DNA and other substances dur-ing the staining of semi-thin sections of epoxyresin with toluidine blue as a rapid stain fortissue.A metachromatic dye visualizes a tissue or cellstructure by staining it a color that is differentfrom the color of the diluted dye solution. Thecolor change is called metachromasy; the cell ortissue element is called the chromotrope. Mostmetachomatic stains are also basic dyes (tolui-dine blue, for example), but this is not an abso-lute rule because some metachromatic dyes areacidic. (However, these are not used to visualizenucleic acids.)

➫Mann–Dominici staining is used to revealDNA metachromatic qualities.

➫Certain staining solutions are mixtures, forexample, the trichroma composed of azorubin(a basic dye), solid green, and naphthol yellow.These solutions are not classified as metachro-matic dyes.

➫A diluted solution of toluidine blue is bluecolored. After staining, nuclei (DNA) are col-ored purple, and cartilage (chondroitin-sulfate)is colored red.


Principles

16

1.2.2.3.2 MECHANISM

A nonaqueous solution of metachromatic dyepreserves the same color whatever the concen-tration or the temperature.

In aqueous solutions, variations of temperatureor concentration modify the color solution. Forexample, solutions of toluidine blue are blue forthe concentrations usually used. They becomepurple, then red if the concentrations areincreased. For the same concentration, the col-oration of the solution is blue at high tempera-ture, and red at low temperature.

1.2.2.3.3 IMPORTANT FACTORS

Tissue reaction depends upon negative charges(or positive in certain cases) that are present onthe chromotrope. To obtain a metachromaticreaction, the tissue charges must be relativelydense (0.5 nm apart). As this distance decreases,metachromasy intensity increases. Conversely,if the distance increases, the tissue color is ortho-chromatic. On the other hand, the increase ofcharge alignment can increase the metachro-matic reaction.

The molecular weight of tissue elements is alsoinvolved: chromotropic substances are generallymolecules with a high molecular weight or mol-ecules that can aggregate themselves to obtain ahigh molecular weight overall.

External factors can modify the metachromaticqualities of a dye. Cations can be competitive tothe basic dye (or anions if the dye is acidic), andthey can lower metachromasy or even stop it.An addition of soluble proteins decreases orstops this phenomenon. However, the use of pro-teolytic enzymes can halt this negative effect.

1.2.2.3.4 CHROMOTROPIC SUBSTANCES

Chromotropic substances are acidic muco-polysaccharides, nucleic acids (especiallyDNA).

➫A spectrometric study of these substanceshas been performed by Lison and Michaelis.When the concentration of aqueous solutionincreases or when temperature decreases, thewavelength for the maximal absorption (λmaxor α band) lowers to the red wavelength and anew absorption wavelength is observed (λ′

maxor β band). Consequently, the color of the solu-tion is displaced from blue to purple or evenred. Passage is observed from the orthochro-matic form (blue) to the metachromatic form(purple or red) for this dye. The explanation isbased on physics and has supplanted other the-ories based on the presence of several molec-ular forms in the same dye solution.

➫This alignment can be increased by hydro-philic or hydrophobic groups. Water mole-cules, which allow polymerization of the dye,can also be used to increase the metachromaticreaction.

➫The higher the valence of competitive ions,the higher the inhibitor effect.

➫Examples are certain lipids and silica parti-cles.



17

1.2.2.3.5 CHOICE OF DYES

The best metachromatic dyes are toluidine blue,azure blue, thionin, and cresyl blue. All are thi-azinic. They are used in aqueous solution; con-centrat ions are 1% (10 g/L) for lesschromotropic substances, 0.1, 0.01, or 0.001%for very chromotropic substances.

1.2.2.3.6 MOUNTING THE SECTION

Mounting is an important factor because themounting medium can inhibit staining.

➫For nucleic acids, mounting with a hydro-phobic medium after dehydration or with ahydrophilic medium without dehydration hasno consequence on staining.

1.2.2.4 Visualization of reductive groups

1.2.2.4.1 REDUCTIVE SUBSTANCES

Numerous substances possess reductive proper-ties, which are used for their histochemicaldetection. Among these substances are cystein,certain pigments, phenolic molecules such ascatecholamine or serotonin, unsaturated lipids,vitamin C, and aldehydes that are produced byreaction of an oxidant with certain chemicalgroups belonging to the sugars or DNA.

1.2.2.4.2 REACTIONS

Four types of reactions are useful to visualize thereductive properties of tissues and cell elements.� Reduction by a silver salt: argent affinity orargyrophily—Reductive substances act on a sil-ver salt to yield a black precipitate that is reducedsilver. Ammonium silver nitrate can be used.

2 AgNO3 + NaOH ➔ Ag2O + NaNO3 + H2OAg2O + 4NH3 + H20 ➔ 2 (Ag(NH3)2OH)

Reaction of silver nitrate with sodium hydroxideyields silver oxide. This last reacts with ammo-nium to yield ammonium silver hydroxide,which is characterized by a diamine ion that willreact with the tissue component.

R–HC=O + 2 (Ag(NH3)OH)➔ RHC=O + 2Ag˚ + 4NH3 + H2O

Other reactions are based on the visualization ofa tissue component by silver deposition, but

➫The reductive properties of aldehydes areused in the Feulgen and Rossenbeck reactionto visualize DNA in photonic microscopy aswell as in electron microscopy.

➫This is a histochemical reaction in which thecell or tissue substance is the reactant. Thissubstance is called argent-affine or reductive-silver. It is visualized as a black deposit ofmetallic silver.

➫Silver impregnation is used to visualize thenucleolar organizer (AgNOR method).


Principles

18

these reactions are not histochemical. They aresilver impregnations, which are often used inhistology. For that, a silver salt is constituted (asin the previous reaction); then it is reduced byformaldehyde. Certain cell or tissue substancesfix the reduced silver by electrostatic binding,adsorption, etc., but these substance are not usedfor the reaction.

2 AgNO3 + NaOH ➔ Ag2O + NaNO3 + H20Ag2O + 4 NH3 + H2O ➔ 2 (Ag(NH3)2OH)or2 AgNO3 + Li2CO3 ➔ Ag2CO3 + 2LiNO2Ag2CO3 + 4NH3 ➔ Ag(NH3)2CO3

� The silver salt can be prepared from sodiumhydroxide or lithium carbonate. After the salt isobtained, it is reduced by formaldehyde.

H2C=O + 2(Ag(NH3)2OH)➔ HCOOH + 2Ag˚ + 4NH3 + H2OorH2C=O + (Ag(NH3)2CO3 ➔ HCOOH + 2Ag˚ + 4NH3 + H2O

� Reduction of ferric ferricyanide—Not used fornucleic acids, reductive groups can be visualizedby reducing ferric ferricyanide to ferric ferrocy-anide, also called Prussian blue. Reductive sub-stances are blue stained. � Other reductive methods (reduction of osmiumtetroxide, tetrazolium salts) can also be used tovisualize reductive groups.

➫These cell or tissue components are calledargentophilic. These reactions are not his-tochemical.

1.2.2.5 Visualization of carbonyl groups

Carbonyl groups are characteristic of aldehydeand ketone, and their visualization is often usedin histochemistry. This visualization can con-cern native groups or groups that have been cre-ated by certain reagents. This last type ofreaction is useful for visualizing DNA.

The visualization can be done in several ways: byreduction of a silver salt, which is linked to thereductive properties of aldehydes and whose reac-tions are argent-affine-like, or by use of the Schiffreagent, which reacts with the carbonyl part of

➫Among these reactions, PAS permits visual-ization of polysaccharides. The Feulgen andRossenbeck reaction permits visualization ofDNA in cell nuclei. This method will bedetailed in Chapter 4 concerning histochemicalmethods.

➫The use of Schiff reagent or a similar reagentallows visualization of aldehydes and ketones.In contrast, use of a method based on reductiveproperties only allows visualization of alde-hydes.



19

a molecular group. Such other methods asdiamine phenylen or hydrazone can also be usedfor DNA detection, but they are less interesting.

1.2.2.6 Quantitative analysis

1.2.2.6.1 METHODS

Several physical methods use quantitative analysisof staining: absorption spectrophotometry, imag-ing quantitative analysis, and flux cytometry. Thisbook only provides some general information.

1.2.2.6.2 HISTOPHOTOMETRY

Histophotometry is used to measure light trans-mission at a precise wavelength, which allowsappreciation of the variations of staining inten-sity as a function of tissue or cell substanceconcentration.

1.2.2.6.3 IMAGING QUANTITATIVE ANALYSIS

It is possible to use automatic imaging quanti-tative analysis methods based on DNA visual-ization for discriminating a same-cell populationinto diploid and polyploid cells. It is essential touse a stoichiometric dye. The Feulgen and Ros-senbeck reaction is often used.

1.2.2.6.4 FLUX CYTOMETRY

The use of flux cytometry has developed over sev-eral years. For this technique, fluorochromes thatare stoichiometrically fixed on DNA are used.

1.2.2.7 Vessels for staining

Staining is done in specific vessels known asBorel’s tubes (Figure 1.9) or in special basketswith grooves in which slides are placed (Figure1.10).

➫If this method is used to appreciate varia-tions of concentrations, it is essential to choosea stoichiometric dye for which different wave-lengths are measured.

Figure 1.9 Borel’s tubes. (From Martoja, R. and Martoja-Pierson, M., Initiation aux techniques de l’histologie animale, 49. With permission.)

Figure 1.10 Basket for slides.(From Mar-toja, R. and Martoja-Pierson, M., Initiation aux techniques de l’histologie animale, 50. With permission.)


Chapter 2

TissuePreparation


Contents

23

Contents

2.1 Sampling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.1.1 Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.1.2 Methods of Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.1.2.1 Tissue Dissection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.1.2.2 Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.2 Fixation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.1 Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.2 General Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.2.2.1 Importance of Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.2.2 Effects of Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.2.3 Chemical Action of Fixatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.4 Different Fixative Types. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.2.4.1 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.4.2 Coagulant Fixatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.4.3 Noncoagulant Fixatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.4.4 Fixative Mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.4.5 Fixative Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.4.6 Nucleic Acid Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.2.5 Chemical Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.5.1 Precautions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.5.2 Fixation by Immersion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.5.3 Fixation by Perfusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.5.4 Fixation by Formalin Vapor . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.5.5 Fixation for Semi-thin and Ultra-thin Sections . . . . . . . . . . . . .

2.2.6 Physical Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.6.1 Cryodesiccation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.6.2 Freezing–Dissolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.6.3 Classic Fixation by Cold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.2.6.4 Chemical and Cold Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.2.7 Holding Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.3 Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.3.1 Paraffin Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.3.1.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.3.1.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.3.1.3 Paraplast Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.3.2 Celloidin Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.3.2.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.3.2.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.3.3 Double Embedding: Celloidin and Paraffin . . . . . . . . . . . . . . . . . . . . .2.3.3.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.3.3.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.3.4 Gelatin Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.3.5 Double Embedding: Agar–Agar and Paraffin . . . . . . . . . . . . . . . . . . .

2.3.5.1 General Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2525252525262626262727282828282930303030313132323333333434343434343535363636373737383838


Tissue Preparation

24

2.3.5.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.3.6 Resin Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.3.6.1 General Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.3.6.2 Epon Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.3.6.3 Durcupant Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.3.7 Treatment and Embedding of Hard Tissues . . . . . . . . . . . . . . . . . . . . .2.3.7.1 Different Hard Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.3.7.2 Noncalcified Hard Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.3.7.3 Calcified Hard Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.4 Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.4.1 Paraffin, Paraffin/Celloidin, or Gelatin/Paraffin Blocks. . . . . . . . . . . .

2.4.1.1 Microtome Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.4.1.2 Cutting the Block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.4.1.3 Difficulties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.4.2 Celloidin Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.4.3 Sections for Plastic Waxes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.4.4 Bone Sections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.4.5 Frozen Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.5 Adhesion of Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.5.1 Paraffin and Double-Embedded Sections. . . . . . . . . . . . . . . . . . . . . . .

2.5.1.1 Water Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.5.1.2 Glycerin–Albumin Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . .2.5.1.3 Adhesion with Gelatinous Water . . . . . . . . . . . . . . . . . . . . . . . .2.5.1.4 Adhesion on Gelatinized Slides . . . . . . . . . . . . . . . . . . . . . . . . .

2.5.2 Adhesion of Collodion Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.5.2.1 General Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.5.2.2 Before Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.5.2.3 After Staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.5.3 Adhesion of Plastic Wax Sections. . . . . . . . . . . . . . . . . . . . . . . . . . . .2.5.4 Adhesion of Frozen Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.5.5 Adhesion of Bone Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.6 Deparaffining and Hydration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.6.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.6.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.7 Collodioning. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.7.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.7.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.8 Smears . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.8.1 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.8.1.1 Smears . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.8.1.2 Imprint . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.8.1.3 Squash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.8.2 Making a Smear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.8.2.1 Dry Blood Smear on Slide . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.8.2.2 Dry Blood Smear on Lamella . . . . . . . . . . . . . . . . . . . . . . . . . .2.8.2.3 Making a Wet Smear . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.9 Cell Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.9.1 Monolayer Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2.9.2 Suspension Cell Culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

38393939414141424243434344454747474748484848494949495050505151515151525252525252525353535353545454


2.1 Sampling

25

2.1 SAMPLING

2.1.1 Types

All material with a biological origin can be iso-lated to visualize its nucleic acids: histologicalsections, smears, karyotypes, and monolayer orsuspension cell cultures.

2.1.2 Methods of Sampling

2.1.2.1 Tissue dissection

Organ or piece dissection before fixation mustbe done with care to preserve the integrity of thetissues and cells for study.

❏

Avoid alteration from autolysis and putrefac-tion by fixing as quickly as possible after thedeath of the organism.

❏

Avoid alteration caused by dissecting toolsby cautious use of the tools.

❏

Avoid osmotic lysis by washing tissues withan iso-osmotic physiological liquid and not withwater.

❏

Avoid tissue drying during dissection byworking in a wet medium.

➫

It is possible to cool the pieces if they cannotbe fixed immediately. However, fixation ofcooled material never yields very good results.

➫

Dissection by means of a razor blade is pre-ferred to dissection with scissors to obtain clearfractures.

2.1.2.2 Cell cultures

2.1.2.2.1

M

ONOLAYER

CELL

CULTURES

Monolayer cell cultures can be cultivated in sev-eral types of flasks. Cell treatment variesdepending on the purpose: to observe entire cellsdirectly upon the culture support or to obtainsemi-thin sections.

❑

Fixation of monolayer cell cultures

1. Empty the cell culture medium.2. Place the fixative.3. Let the cell remain in contact with the fix-ative for several hours.

➫

The flask types used in monolayer cell cul-ture are variable: Rous boxes, microplates,Leighton tubes containing a detachable slideon which cell culture occurs, or “Labtek”which is a culture box whose bottom can bedetached to observe the cells directly with amicroscope.

➫

All fixatives can be used whatever the celltype.

➫

Fixation duration depends on the cell typeand the fixative. Because of the thinness ofmonolayer cell cultures, this duration can bevery short (15 min, for example).


Tissue Preparation

26

❑

Preparation of cell culture to obtain semi-thin sections.

1. Empty the cell culture medium.2. Cover the monolayer with 0.25% trypsin,at 37˚C.3. Observe the evolution of cells quicklywith a reversed microscope.4. Incubate at 37˚C.5. When the cell culture begins to slip, stopthe trypsin action with cell culture medium.6. Dissociate cell masses by aspirating andplunging back the cell suspension (20 to 30times).7. Centrifuge the cell suspension at 600

g

for10 min.8. Decant and rinse the bottom by addingsuspension to the fixative.

2.1.2.2.2

S

USPENSION

CELL

CULTURES

1. Remove cells from the vessel.2. Centrifuge the suspension at 600

g

for 10min.3. Decant and rinse the bottom by addingsuspension in the fixative.

➫

Trypsin must be used with care

. If use isprolonged, plasmic membranes and eventuallycells will be destroyed.

➫

Centrifugation must be done with care toavoid mechanical destruction of cells.

➫

Centrifugation must be done with care toavoid mechanical destruction of cells.

2.2 FIXATION

2.2.1 Definition

Fixation (or preservation) consists of preparingan organism, an organ, a tissue, or even a cellto be held, in death, in a state as close as possibleto the living state.

2.2.2 General Principles

2.2.2.1 Importance of fixation

In every histological operation, fixation is the firststage, one that is indispensable for good preserva-tion of cell and tissue components. These compo-nents must remain in a state as close as possible tothe life state. Fixation must be performed in a waythat allows visualization of these components byhistological or histochemical methods. The process


2.2 Fixation

27

must not eliminate the components, and it mustnot react with the atomic groups that will be usedto visualize the structure. Fixation must preservethe tissue morphology in a recognizable form,which is essential to good localization of the mol-ecules of interest.

2.2.2.2 Effects of fixation

The effects of fixation on the tissues are numerous:

❏

Immobilization of cell components afterextraction from the natural environmentalmedium

❏

Inhibition of cell autolysis that is due toenzyme liberation after degradation of lyso-somic membranes

❏

Inhibition of putrefaction due to microorgan-isms

❏

Tissue hardening

❏

Modification of tissue refraction index thatpermits observation of them even before stain-ing

❏

Rendering insoluble cell substances

❏

Modification of tissue volume as a functionof the fixative

❏

Effects on dye affinity

➫

If hardening is reasonable and permits oneto obtain histological sections, the fixative iscalled “tolerant.” If hardening is too great anddoes not permit one to obtain sections, thefixative is called “intolerant.”

➫

Modification of cell volume after fixativeaction must be considered, especially for mor-phometric studies. Using the same fixative toobtain comparative results is recommended.

➫

Acetic acid, for example, separates proteinsfrom nucleic acids, which can lead to theextraction of the latter. Use of formalin oftendecreases the intensity of certain stains.

2.2.3 Chemical Action of Fixatives

A good histological fixative is a good proteinfixative because the cell or tissue structure islinked to these macromolecules. Chemical fixa-tives react on reactive protein groups by estab-lishing bridges between these molecules.Formaldehyde (H

2

C=O) reacts with amines,imines, guanidyls, hydroxyls, sulfhydryls, car-boxyls, peptic binding, and aromatic nuclei. Itestablishes methylene bridges between proteinmolecules.

➫

At room temperature, formaldehyde is a gas.Formalin, which is usually used, is a solutionof formaldehyde in water. Trade formalin, soldat 30 or 40% in water, is used in diluted solu-tion:

�

Formalin

10 mL

�

Water

100 mL


Tissue Preparation

28

2.2.4 Different Fixative Types

2.2.4.1 Definitions

Fixatives can be simple, often consisting of onlyone molecule. They also can be constituted of amixture of simple fixatives. Among simple fix-atives, certain are coagulant or denaturant. Theiraction induces a protein denaturation that lendsa reticulated aspect to cytoplasm. Other fixativesare not coagulant and the aspect of cytoplasm ishomogeneous.

2.2.4.2 Coagulant fixatives

2.2.4.2.1

E

THANOL

Ethanol (70 to 100%) is essentially used to pre-serve mineral elements. It is also used as a com-ponent of fixative mixtures (Carnoy’s fluid, forexample) used for protein and nucleic acid his-tochemistry. Tissues are hardened with its use.

2.2.4.2.2

P

ICRIC

ACID

Picric acid is also a coagulant fixative used asan adjuvant in fixative mixtures (Bouin’s fluid,Halmi’s fluid). Its yellow color does not interferewith staining sections with dyes.

2.2.4.2.3

M

ERCURY

CHLORIDE

Mercury chloride (or sublimate) HgCl

2

is alsoused in several mixtures. It penetrates quickly butnot as deeply. Tissues are hardened by its use. Itcan also cause a precipitate on the sections.

➫

Ethanol is a good fixative used for methodsbased on detection of carbonyls obtained afteracidic hydrolysis.

➫

If the yellow color of a section obtained fromtissue fixed by a fluid containing picric acid iscumbersome, the color can be eliminatedbefore section staining by using alkaline baths,such as lithium carbonate. Be careful to sepa-rate the sections. The color can also be elimi-nated by use of 70% ethanol or running water.

➫

Mercury chloride is very corrosive

. Avoidcontact with metal dissecting tools.

➫

It is possible to eliminate mercury chlorideby rinsing sections with lugol, then withsodium hyposulfite.

2.2.4.3 Noncoagulant fixatives

2.2.4.3.1

F

ORMALDEHYDE

Formaldehyde can be used alone or in a mixedfixative. If used alone, it is often helpful to neu-tralize or buffer it to avoid formation of formicacid by oxidization. Formaldehyde, which isused as formalin, penetrates quickly into tissuesthat are not excessively hardened. This fixativepermits a relatively lengthy preservation of tis-sues, but it can modify staining qualities.


2.2 Fixation

29

2.2.4.3.2

O

SMIUM

TETROXIDE

Osmium tetroxide is a very good cytologicalfixative that reacts with lipids and phospholipidsthat belong to the cell membrane structure. Itpermits cell proofing. It is also a powerful oxi-dant that must be avoided in histochemical reac-tions. Osmium tetroxide penetrates very little intissues that are hardened, making it impossibleto obtain sections. It is also used to fix smears.

2.2.4.3.3

A

CETIC

ACID

Acetic acid is also used in fixative mixures. Itquickly penetrates into the tissues and stabilizesnucleoproteins. If it is used at too high a con-centration, it can separate nucleic acids andnucleoproteins. It is a good fixative for thenucleus if it is used at a concentration between0.3 and 5 mL in 100 mL of fixative.

➫

Osmium tetroxide is also called osmic acidby histologists, but this name is chemicallyincorrect because this molecule has no acidiccharacteristics.

➫

Osmium tetroxide is also a good fixative foruse in electron microscopy.

2.2.4.4 Fixative mixtures

Fixative mixtures are most often used. Theeffects of several simple fixatives are additive.Below are listed the fixative mixtures most oftenused for histochemistry and histology. They canbe used to visualize nuclei and nucleic acids.

�

Alcohol–Formalin

�

Baker’s Fluid

�

Bouin’s Fluid

�

Bouin–Hollande’s Fluid

�

Carnoy’s Fluid

�

Flemming’s Fluid

�

Formalin–Calcium

�

Neutral Formalin

➫

See

Chapter 7: Preparation of Products.

➫

See


➫

See


➫

See


➫

See


➫

Must be prepared at time of use.

➫

See

Chapter 7: Preparation of Products

➫

Must be prepared at time of use.

➫

See

Baker’s fluid.

➫

See


➫

See



Tissue Preparation

30

�

Salt Formalin

�

Buffered Formalin

�

Halmi’s Fluid

�

Helly’s Fluid

� Heidenhain Susa

� Zenker’s Fluid

➫See Chapter 7: Preparation of Products

➫0.1 M, pH 7.➫See Chapter 7: Preparation of Products


➫Helly’s fluid is also called Zenker formalin.➫See Chapter 7: Preparation of Products



2.2.4.5 Fixative duration

� Bouin’s fluid 24 to 48 h� Carnoy’s fluid 4 h� Formalin indefinitely

➫The duration must be adapted to the thick-ness of the sample (5 to 10 mm for photonicmicroscopy).➫To improve the fixation of large pieces, pre-fixing can be done for 24 to 48 h in formalin,before post fixation with Bouin’s fluid for 1 to24 h.

2.2.4.6 Nucleic acid fixation

Nucleic acids are linked to proteins, and fixa-tives that act on proteins are suitable for nucleicacids. Histological or histochemical visualiza-tion can be performed on tissues fixed by meansof classic fixatives. But certain histochemicalreactions that are used to visualize DNA, suchas the Feulgen and Rossenbeck reaction, are sen-sitive to the acidic character of the fixative.These reactions need experimental conditionsthat differ according to the pH of the fixative. Ifthe fixative is used without any adaptation, DNAvisualization can become impossible.

➫Conservation of cytoplasmic RNA is moredifficult than of DNA. RNA is linked to ribo-somes that have an important phospholipidicfraction that is very sensitive to the fixative.The use of solvent as a fixative must beavoided. On the other hand, dehydration afterpreservation has no detectable effect.

2.2.5 Chemical Fixation

2.2.5.1 Precautions

Whatever the fixative and the cell or tissue ele-ment that is to be visualized, the fixation mustanswer to several imperatives, and several pre-cautions must be taken.


2.2 Fixation

31

❏ Avoid getting blood or mucus on the tissuesurface. These biological elements may becomehard after coagulation or they may polymerizeand constrict fixative penetration.❏ Use a sufficient volume of fixative.❏ If fixation is to be of long duration, exchangethe fixative periodically with a new solution. Anequilibrium is established between intra- andextra-tissue fluids and the fixative. In the end,the fixative will not penetrate, and tissue and cellcomponents will not be correctly immobilized.❏ Use relatively wide bottles. Pieces must notbe allowed to stick to the bottle wall above thefixative level.

➫The presence of blood on the surface of tis-sues also causes blood cells (leukocytes, redblood cells) to be carried to areas where theyare not usually present.

➫One method is to enclose the piece in a bagof gauze and hang it from the mouth of thebottle.

2.2.5.2 Fixation by immersion

To fix by immersion, immerse the tissue frag-ment in the fixative as quickly as possible.

➫To avoid autolysis and putrefaction, theorgan or tissue fragment must be extracted veryquickly after the animal’s (or vegetation’s)death.➫Do not forget to label each fragment.

2.2.5.3 Fixation by perfusion

This method is used for the fixation of tissuesthat are particularly fragile. The whole animalcan be subjected to the perfusion.

To perform a perfusion (Figure 2.1):1. Anesthetize the animal.2. Open the thoracic cage.3. Remove the pericardium.4. Incise the left ventricle near the top of theheart.5. Insert the nozzle into the aorta.6. Hold the nozzle with a Mohr grip.

➫Fixation by perfusion can be used for brain,for example.

1 = Open thoracic cage (not shown in thefigure).2 = Incise left ventral ventricle.3 = Introduce the nozzle.4 = Clamp the nozzle by means of a Mohr’sgrip.5 = Open the right auricle.

Figure 2.1 Perfusion. (From Morel, G.,Hybridation in Situ, Polytechnica, EditionsEconomica, 1998, 67. With permission.)

2

5

4

3


Tissue Preparation

32

7. Open the right auricle.8. Rinse with physiological buffer.9. Perfuse with fixative.10. After perfusion, dissect the organs andplunge them into fixative.

2.2.5 4 Fixation by formalin vapor

This method is used to fix a blood smear.1. Place the fixative in the bottom of a bottle.2. Close the bottle.3. Quickly place the smear above the fixativefor several seconds.

➫2% osmium tetroxide in distilled water isusually used.

➫It is obvious that the face of the slide withthe smear is placed above the fixative.

Fixation can take from 10 s to 3 min.

2.2.5.5 Fixation for semi-thin and ultra-thinsections

2.2.5.5.1 PRINCIPLE

Semi-thin sections are often used to reduce thetissue before obtaining ultra-thin sections forelectron microscope observation. For this, fixa-tion must be very precise to avoid unsatisfactoryimages. The fixative must be iso-osmotic to tis-sue fluids; it must be at the same pH; and it mustpossess the same ionic composition.To accomplish this, the tissue is generally firstpreserved in a mixture of formaldehyde and glu-taraldehyde to polymerize proteins. Then it mustbe postfixed by osmium tetroxide to imperme-abilize the cell membranes by reaction with thephospholipidic bilayer.One method for fixation is given here.

2.2.5.5.2 GLUTARALDEHYDE/PARAFORMALDEHYDE FIXATIVE

❑ Sorensen buffer (PBS) 0.2 M, pH 7.4� Monosodium phosphate (solution 1)—Monosodium phosphate 3.12 g—Distilled water 100 mL� Disodium phosphate (solution 2)—Disodium phosphate 7.16 g

➫Other fixative preparation modes for elec-tron microscopy exist, especially those usinganother buffer (i.e., cacodylate buffer). Theycan also be used for tissue preparation beforevisualizing nucleic acid on semi-thin sections.It is also possible to use only paraformalde-hyde alone or glutaraldehyde alone.


2.2 Fixation

33

—Distilled water 100 mL� Buffer—Solution 1 19 mL—Solution 2 81 mL

❑ Glutaraldehyde� Glutaraldehyde 4 g� Distilled water 100 mL

❑ Paraformaldehyde� Stock solution—Paraformaldehyde 4 g—Distilled water 10 mL� Working solution—Stock solution 2 g—Distilled water 100 mL

❑ Preparation of fixative� Glutaraldehyde 4% 100 mL� Paraformaldehyde 2% 100 mL� Buffer 0.2 M, pH 7.4 200 mL

2.2.5.5.3 OSMIUM TETROXIDE

� Stock solution—Osmium tetroxide 0.5 g—Distilled water 25 mL� Working solution—Stock solution 10 mL—Buffer 0.2 M, pH 7.4 10 mL

➫Glutaraldehyde is prepared from a solutionalready diluted to 25 or 50%.➫To prepare

— Dissolve paraformaldehyde in tepidwater (about 3 min)— Heat for 20 min at 80°C— Add sodium hydroxide (2 drops)

➫Fixative can be stored for several days at4°C.

➫Osmium tetroxide is available in an ampoulecontaining 0.5 g.

2.2.6 Physical Fixation

In addition to chemical fixation by means ofmolecules that react with the tissue component,there are methods that are based exclusively onphysical principles.

2.2.6.1 CryodesiccationIn cryodesiccation a fresh tissue is frozenquickly, then dried under vacuum at a very lowtemperature. Tissue water goes directly from thesolid state to the gaseous state. The tissue frag-ment is then directly embedded in melted wax.

➫Cryodesiccation has a lyophilization phasethat avoids tissue component diffusion. Tissuemolecules do not undergo any chemical mod-ification.

2.2.6.2 Freezing–dissolutionIn freezing–dissolution a fresh tissue is frozenquickly, then the ice is dissolved with absoluteethanol. Embedding is then done in melted wax.


Tissue Preparation

34

2.2.6.3 Classic fixation by coldIn certain cases, fresh tissue is directly fixed bycold, then cut with a freezing microtome or acryotome.

2.2.6.4 Chemical and cold fixationTissues that were fixed by chemical fixative maybe cut without embedding by means of a freez-ing microtome or a cryotome.

2.2.7 Holding Fluids

Preserved tissues can be stored in holding fluids:❏ Bouin’s fluid preservation 70% ethanol❏ Formalin preservation Formalin❏ Carnoy’s fluid preservation Butanol

➫Ethanol must be avoided if lipids are to bevisualized.➫Butanol must be avoided if lipids are to bevisualized.

2.3 EMBEDDING

2.3.1 Paraffin Embedding

2.3.1.1 Principle

Paraffins are insoluble in water, so it is not pos-sible to immerse a tissue containing waterdirectly in paraffin. The tissue must first be dehy-drated by ethanol (or acetone) to a greater andgreater degree. However, paraffin is also not sol-uble in ethanol or acetone, so the tissue must beimmersed in an intermediate substance that issoluble in the solvent and in paraffin. Amongthese substances are toluene, xylene, chloro-form, and warm butanol. This operation is calledclarification because tissue fragments becometransparent.The prepared tissue is then immersed in liquidparaffin for several hours. This is the impregna-tion stage. After changing the paraffin bath, theorgan is immersed in liquid paraffin that is con-tained in a receptacle at room temperature.When the paraffin is solid, the embedding isfinished and the organ can be sectioned with amicrotome.

➫Paraffins, from Latin parum affinis (littlereactive), are alcanes. They are organic mole-cules exclusively constituted of carbon andhydrogen with no functional group that canreact with cell or tissue molecules. Therefore,hard blocks can be obtained, allowing them tobe cut.

➫Receptacles include Leuckart’s bars (Figure2.2) or cassettes.


2.3 Embedding

35

➫Leuckart’s bars are arranged to form a recep-tacle into which liquid paraffin is poured. Thetissue piece is immersed in the paraffin. Afterseveral minutes, a solid block forms that isready to be cut.

Do not forget to label each block.

Figure 2.2 Leuckart’s bars for embedding. (From Morel, G., Hybridation in Situ, Poly-technica, Editions Economica, 1998, 81. With permission.)

2.3.1.2 Protocol

❑ Dehydration� Pieces preserved by Bouin’s fluid or formalin

1. Ethanol 70% 4 h2. Ethanol 96% 2 × 12 h3. Ethanol 100% 2 × 4 h

� Organs that are preserved in Carnoy’s fluid aredirectly immersed in butanol.❑ Clarification

4. Immerse in butanol 2 × 12 h

❑ Paraffin impregnation5. Leave the tissue in melted paraffin for 4 to12 h depending on the tissue type: 4 h forliver, kidney, spleen, and lung, and 12 h forother tissues. The temperature used is themelting point of paraffin.

❑ Embedding6. Embed the impregnated tissue in a paraffinblock that is formed with a mold (Leuckart’sbars, embedding cases, etc.). The block isready for cutting.

➫The duration of baths in the different ethanolsolutions can be increased: 24 h for each bathin ethanol 95% and 4 h in each bath in absolute(100%) ethanol. Conversely, in certain cases,these baths can be decreased (only 1 h in eachbath, but at 40˚C).➫The duration of the butanol bath can also bemodified. A lengthened stay that can reach 24 hand more is useful for embedding. Butanol canalso allow preservation of tissue fragmentsbefore embedding.

➫In the case of a manipulation error, it issometimes useful to embed the pieces again,by immersing the cut block in melted paraffin.When the piece is melted out of its solid par-affin encasement, remake the block.

2.3.1.3 Paraplast embedding

2.3.1.3.1 PRINCIPLE

Paraplast is a mixture of natural paraffins andsynthetic polymers. It has very good qualities ofresistance and elasticity. The embedding tech-nique is similar to that employed with paraffin.

2.3.1.3.2 PROTOCOL

❑ Dehydration� Pieces preserved in Bouin’s fluid or formalin

➫It is possible to obtain thinner sections withparaplast than with paraffin. It also allows veryhard tissues or organs with parts of varyinghardness to be cut.


Tissue Preparation

36



4. Immerse in butanol 2 × 12 h

❑ Paraplast impregnation5. Leave the tissue in melted paraplast for 4 to12 h, depending on the tissue type: 4 h forliver, kidney, spleen, and lung, and 12 h forother tissues. The temperature used is themelting point of paraplast.

❑ Embedding6. The impregnated tissue is embedded inparaplast that is formed with a mold (Leuck-art bars, embedding cases, etc.).

The block is ready for cutting.

➫See Section 2.3.1.2. The duration of baths inthe different ethanol solutions can be increased:24 h for each bath in ethanol 95% and 4 h ineach bath in absolute (100%) ethanol. Con-versely, in certain cases, these baths can bedecreased (only 1 h in each bath but at 40˚C).➫See Section 2.3.1.2. The duration of thebutanol bath can also be modified. A length-ened stay that can reach 24 h or more is usefulfor embedding. Butanol can also allow preser-vation of tissue fragments before embedding.

➫In case of a manipulation error, it is some-times useful to embed the pieces again byimmersing the cut block in melted paraplast.When the piece is melted out of its solid para-plast encasement, remake the block.

2.3.2 Celloidin Embedding

2.3.2 1 Principle

Celloidin (nitrocellulose) embedding is a sim-ple technique that does not require any warm-ing. Like paraplast, this embedding mediumpossesses a rigid and elastic consistency thatallows hard tissues or tissues with varying con-sistency to be cut into thin sections. It alsoallows embedding and sectioning of very largepieces.

2.3.2.2 Protocol



� Organs that are preserved in Carnoy’s fluid aredirectly immersed in butanol.

➫Celloidin embedding has been widely usedto study eyes, which are always difficult to cutbecause of the presence of both a hard structure(the crystalline lens) and smooth structures(vitreous and aqueous humor). This mediumhas allowed the embedding of such largeorgans as human brain.➫Dehydration must be very carefully done.

➫However, celloidin embedding is a slowmethod that requires several weeks. It does notpermit sections with a thickness less than 10µm to be cut. It is also difficult to obtain serialsections with this technique. Another inconve-nience is the necessity to store blocks in 70%ethanol.


2.3 Embedding

37

❑ Embedding4. Immerse in ethanol/ether. 24 h5. Immerse in 2% celloidin in ethanol/ether 1:1

7 days6. Immerse in 4% celloidin in ethanol/ether 1:1

7 days7. Immerse in 7% celloidin in ethanol/ether 1:1

7 days8. Permit the ethanol/ether mixture to evapo-rate under a vacuum bell containing a cupwith sulfuric acid.

9. Steam with ethanol 70% or formalin

10. Immerse in Bolles–Lee’s fluid. 5 min

11. Store blocks in 70% ethanol.

➫Organ embedding is done in a paper or card-board mold.

➫The embedding mixture contains 100% eth-anol and ethyl ether.➫Sulfuric acid is a powerful dehydratingagent.

➫Ethanol or formalin vapors are obtained byputting the preparation under a vacuum bell inwhich formalin or ethanol is present. Thisoperation hardens the block.➫Bolles–Lee’s fluid is used for clarifying theblock. Composition of Bolles–Lee’s fluid:

� Chloroform 100 mL� Cedar oil 200 mL

2.3.3 Double Embedding: Celloidin and Paraffin

2.3.3.1 Principle

This method is used for histological treatmentof organs or entire small animals that containnumerous small holes.Preserved and dehydrated pieces are impregnatedwith celloidin, then with paraffin. Celloidin entersthe holes that paraffin cannot reach. Subsequentencasement with paraffin then forms a block withall the classic qualities of paraffin.

➫This method is especially useful for insects,small crustaceans, and lungs.

2.3.3.2 Protocol

❑ Dehydration� Pieces preserved by Bouin’s fluid or formaline

1. Ethanol 70% 4 h2. Ethanol 96% 2 × 12 h3. Ethanol 100% 2 × 4 h4. Acetone (propanone) 2 × 30 min

� Organs that are preserved in Carnoy’s fluid aredirectly immersed in butanol.❑ Celloidin impregnation

5. Add methyl salicylate until pieces fall tothe bottom of the receptacle.


Tissue Preparation

38

6. Add celloidin 1% in methyl salicylate untilpieces fall to the bottom of the receptacle.

❑ Paraffin impregnation7. Immerse pieces directly in a liquid paraffinbath for 4 to 12 h, as in normal paraffinembedding.

❑ Embedding8. Embed pieces in liquid paraffin that is con-tained in a mold. After cooling, a block isformed, as after normal paraffin embedding.

2.3.4 Gelatin Embedding

This type of embedding is used for fragile orsmall objects. The preserved fragments areimmersed in lukewarm gelatin solutions atincreasing concentrations (10%, then 20% gel-atin in water). The solution and fragments arethen placed into a mold and hardened withformalin.

➫Gelatin embedding also allows one to obtainfrozen sections.

2.3.5 Double Embedding: Agar–Agar and Paraffin

2.3.5.1 General principle

This type of embedding is used to cut smalltissue fragments at ambient temperature that aredifficult to embed in a classic paraffin block.This embedding has all the advantages of normalparaffin embedding. In the technique, preservedand dehydrated tissue is first embedded withgelose, and then with paraffin.

➫Agar–agar and paraffin double embedding isparticularly useful for oocytes or small eggsand for embedding pituitary organs, whichmust be sectioned according to an orienteddraft.

2.3.5.2 Protocol

❑ Reagents� Agar–agar gel

1. Add Agar–agar. 1.3 g2. Rinse with boiling distilled water. 100 mL3. Let agar–agar dissolve.4. Add formalin 2.5%.

� Paraffin❑ Agar–agar embedding

1. Spread a 1- to 2-mm-thick agar–agar layer.2. Place the pieces to be embedded on the layer.3. Cover the pieces with melted agar–agar.


2.3 Embedding

39

4. Let cool.5. Immerse in ethanol 70%. 30 min6. Cut the block.




4. Immerse in butanol. 2 × 12 h❑ Paraffin impregnation

5. Leave the tissue in melted paraffin for 4 to12 h as a function of the tissue type: 4 h forliver, kidney, spleen, and lung, and 12 h forother tissues. The temperature used is themelting point of paraffin.

❑ Embedding6. Embed the impregnated tissue in a paraffinblock that is formed with a mold (Leuckart’sbars, embedding cases, etc.).


➫See Section 2.3.1.2. The duration of thebaths in the different ethanol solutions can beincreased: 24 h for each bath in ethanol 95%and 4 h in each absolute (100%) ethanol bath.Conversely, in certain cases, these baths can bedecreased (only 1 h in each bath, but at 40˚C).➫See Section 2.3.1.2. The duration of thebutanol bath can also be modified. A length-ened stay that can reach 24 h or more is usefulfor embedding. Butanol can also allow preser-vation of tissue fragments before embedding.

➫See Section 2.3.1.2. In case of a manipula-tion error, it is sometimes useful to embed thepieces again. For that, immerse the cut blockin melted paraffin. When the piece is meltedout of its solid paraffin encasement, remake theblock.

2.3.6 Resin Embedding


Resin embedding is used primarily for electronmicroscopy, but can also be used for photonicmicroscopy. Blocks that are formed are particularlyhard, allowing one to obtain semi-thin sections (0.5to 1 µm) and to increase the precision of images.

2.3.6.2 Epon embedding

2.3.6.2.1 PRINCIPLE

In certain cases (electron microscopy, semi-thinsections), it is necessary to make blocks in a hardresin that allows very thin sections (0.5 to 1 µm)to be cut. For that, one can use epoxy waxes thatpolymerize at about 60˚C. Epon is a syntheticpolymer that comprises several monomer mole-cules, which are associated by a cross-linker. Poly-merization is performed with a catalyst. By usingseveral monomer proportions, one can obtainblocks of several hardnesses.

➫It is necessary to use a microtome equippedwith a glass knife. These waxes cannot be sec-tioned with a steel razor without damage.


Tissue Preparation

40

2.3.6.2.2 PROTOCOL

❑ Reagents� Epikote 812� DDSA� MNA� DMP30� Propylene oxide

❑ Dehydration1. Ethanol 30% 10 min2. Ethanol 50% 10 min3. Ethanol 70% 2 × 10 min4. Ethanol 95% 10 min5. Propylene oxide (4˚C) 10 min

❑ Quick dehydration—Ethanol 70% 2 × 10 min—Ethanol 95% 2 × 10 min—Ethanol 100% 2 × 10 min—Propylene oxide (4˚C) 10 min

❑ Impregnation and embedding medium� Epon A—Epikote 812 31 mL—DDSA 50 mL� Epon B—Epikote 812 50 mL—MNA 44 mL� Embedding medium—Epon A 40 mL—Epon B 60 mL—DMP 30 1.7 mL

❑ Substitution medium� Embedding medium 50 mL� Propylene oxide 50 mL

❑ Substitution6. Substitution medium 1 h

at RT❑ Impregnation

7. Impregnation medium 12 hat RT

❑ Embedding8. Embed in molds with different forms:

� Embedding medium 2 hat 37˚C

� Embedding medium 3 daysat 60˚C

➫The dehydration stage is indispensablebecause waxes are not hydrosoluble. The pro-cess must be performed particularly quickly topreserve cell and tissue structures.

➫Propylene oxide, also called 1,2-epoxy-pro-pane, is a solvent for embedding wax.

➫Work under a hood. Avoid contact withskin.

➫Epon A and B proportions can be modifiedaccording to the hardness that is desired forthe block. For a soft block, increase the EponB proportion. To obtain a hard block, increasethe Epon A proportion.

RT = room temperature.

➫Fill the mold with embedding medium andembed the object to be studied. Do not forgetto label.


2.3 Embedding

41

2.3.6.3 Durcupant embedding

❑ Reagents� Durcupant CY 212� Durcupant HY 964� Dibutyl phthalate� Accelerator DY 064

❑ Dehydration1. Ethanol 70% 2 × 15 min2. Ethanol 90% 2 × 15 min3. Ethanol 95% 2 × 15 min4. Ethanol 100% 2 × 20 min

❑ Impregnation and embedding medium� Durcupant I—Durcupant CY 212 15 mL—Hardener HY 964 5 mL—Dibutyl phthalate 1 mL� Durcupant II—Durcupant CY 212 10 mL—Hardener HY 964 10 mL—Dibutyl phthalate 1 mL� Embedding medium—Durcupant II 21 mL—Accelerator DY 064 0.35 mL� Impregnation medium—Durcupant I 50 mL—Ethanol 100% 50 mL

❑ Impregnation5. Impregnation medium 30 min6. Durcupant I 12 h

at 60˚C7. Durcupant II 24 h

at 60˚C❑ Embedding

8. Embed in molds with different forms:� Embedding medium 72 h

at 20˚C

➫Durcupant is also called “araldite.”

➫The dehydration stage is indispensablebecause waxes are not hydrosoluble. It mustbe performed particularly quickly to preservecell and tissue structures.

➫See Section 2.3.6.2.2. Fill the mold withembedding medium and embed the object tobe studied. Do not forget to label.

2.3.7 Treatment and Embedding of Hard Tissues

2.3.7.1 Different hard tissues

There are two categories of hard tissues. The hard-ness of the first is linked to scleroproteins Thehardness of the second is linked to calcium. Tissuetreatment will vary according to the cell or tissuecomponent that is responsible for the hardness.

➫An example is the chitin of insects or crus-taceans.➫Bones are an example.


Tissue Preparation

42

2.3.7.2 Noncalcified hard tissues

The embedding process is similar to that forstandard soft tissue. To improve the inclusionconsistency and to facilitate sectioning, one canuse clarifying agents that are not hardeners, suchas amyl acetate or cedar oil. Paraffin baths canbe lengthened.

2.3.7.3 Calcified hard tissues

Histological study of calcified tissues can bedone from decalcified preparations, but this isnot a general rule.

2.3.7.3.1 DECALCIFICATION

Decalcifying agents can be used on preservedtissues, but never on fresh tissues. These agentscan be included in the fixative, or they can beused after preservation.

1. Decalcifying agents:a. Nitric acid 5 to 7.5% in water, alone orwith an ethanol–formalin fixativeb. Hydrochloric acid 4 to 8% in waterc. Citric acid 7% in waterd. Sulfosalicylic acid 6 to 8% in watere. Trichloracetic acid

2. It is also possible to use chelating agentssuch as EDTA or solutions such as RDO.3. It is also possible to use an ion exchangeresin or electrophoresis.4. The decalcifying effect is stopped whendecalcification is judged sufficient.

5. Preparation of calcified tissues:a. Thin slices can be cut with a band saw;then they are placed on a glass plate withan abrasive paste.b. Tissues can be cut with a freezingmicrotome.c. Inclusion can be done with a specialwax.

➫See Section 2.2.

➫The easiest way to appreciate decalcificationis to push a pin into the tissue to test its hard-ness. One can also detect calcium in the fixa-tive by use of a chelator that provides a coloredreaction. Radiography is a more elaboratemeans, but it is also the least useful.

➫This method is used to study bones, eventhough the sections are thick.

➫See below.


2.4 Sections

43

2.3.7.3.2 BONE EMBEDDING

1. Fixation—All fixatives can be used unlessthey contain a decalcifying substance, i.e., anacidic element.2. Dehydration and substitution

a. Ethanol 100% 48 hb. Toluene 48 h

3. Impregnation and embedding—Methylmethacrylate (MMA) is used. It is polymer-ized by use of benzoyl peroxide.

c. MMA 1—MMA 100 mL—Dibutyl phthalate 25 mLd. MMA 2—MMA 1 100 mL—Benzoyl peroxide 1 mLe. MMA 3—MMA 1 100 mL—Benzoyl peroxide 2.5 mL

4. Protocolf. MMA 1 48 hg. MMA 2 48 hh. MMA 3 48 h

5. Embeddingi. MMA 3 72 h

at 37˚C➫Blocks are transparent.

2.4 SECTIONS

2.4.1 Paraffin, Paraffin/Celloidin, or Gelatin/Paraffin Blocks

2.4.1.1 Microtome sections

Blocks are placed on the stage of a verticalmicrotome (Minot’s microtome; Figure 2.3).The block can be positioned on the stage inseveral manners. In the modern microtomes, theencased material is placed on the stage with apair of clamps.In other microtome types, clamps can be usedto grip the block of wax.Sections are usually cut 4 to 7 µm thick. Sectionthickness is ranged on the microtome. On mod-ern types, the thickness is given in micrometers.

➫In older microtomes, the paraffin block isstuck to the stage by warming the stage withan alcohol lamp and applying it against theparaffin block.

➫In older models, the thickness is given inround fractions of the endless screw that con-stitutes the forward movement system of themicrotome.


Tissue Preparation

44

The block is submitted to a vertical up-and-down motion. When it goes down, a section iscut by a steel razor. The block then goes upbehind the blade.

Sections can be directly removed from the bladewith a brush. In certain microtome types, theycan be removed onto a belt conveyor.Generally, the microtome movement is manipu-lated by hand. This allows the histologist to varythe cutting speed.

➫The blade can be straight (for dense and hardtissues) or can have a concave face in front ofthe outer side (for soft and less dense tissues).

➫Avoid steel objects, such as scalpels, for-ceps, etc.

➫Some microtomes are automatic.

Figure 2.3 Microtome.

2.4.1.2 Cutting the block

Before the paraffin block is placed on the stage,it must be cut (Figure 2.4). The paraffin aroundthe object to be sectioned must be removed,leaving the object enclosed in a trapezoidal par-affin block. The lower and upper sides of theblock must be parallel.

➫During cutting, the lower side of the secondsection sticks to the upper side of the firstsection (Figure 2.4). If the two sides are notparallel, the resulting ribbon of sections willbe curved, which can make it difficult to mounta series of sections on a slide.

Figure 2.4 Cutting the block.

Figure 2.5 Illustration of ribbon of sections cut with a microtome. (From Martoja, R. and Martoja-Pierson, M., Initiation aux techniques de l’histologie animale, 43. With permission.)

43X01


2.4 Sections

45

2.4.1.3 Difficulties

Several difficulties occasionally occur during thecutting operation. Some problems, their causes,and their solutions are given below.

2.4.1.3.1 RIBBON IS NOT FORMED

� Blade is blunt.

� Sections are thick.

� Paraffin is too hard.

� Room temperature is too low.

� Angle between object and blade is too open.� Other reasons.

2.4.1.3.2 RIBBON IS CURVED

� Upper and lower sides are not parallel.� Upper and lower sides are not parallel to

the razor edge.� Razor edge is irregular or blunt.

� Block sides are not at the same temperaturebecause of light, a warming source, and soon that give different hardnesses to the wax.

2.4.1.3.3 THICKNESSES OF SECTIONS ARE DIFFERENT

� Angle between specimen and blade is toosmall, section cannot be obtained.

� Angle between razor and object is too great.� Microtome vibrates.� Section is too hard.

� Wax is too soft.

2.4.1.3.4 SECTIONS ARE COMPRESSED

� Razor edge is blunt.

� Temperature is too high.

� Angle between specimen and blade is toosmall.

� Section speed is too great.� Tissue fragment is compressed but not the wax.

➫Displace the blade laterally.➫Replace the blade.➫Reduce the thickness of the section bymanipulating the advance of the microtome.➫Reembed by melting the paraffin andembedding the object in a new wax with alower melting point.➫Warm the blade and the specimen with alamp, or dip the specimen into tepid water, orblow on the specimen (in this case, verify theobject is locked in the higher position).➫Change the blade pitch.➫Hold sections with a thin brush as they areformed.

➫Cut the block again to obtain parallel sides.➫Change the block position.

➫Displace the razor laterally.➫Use another razor.

➫Put the microtome where it is not subject totemperature variations.

➫Change the blade pitch.

➫Change the blade pitch.➫Check the screw.➫Immerse the block in water; this will makeit soft.➫Embed the specimen again with a paraffinhaving a higher melting point.

➫Displace the razor laterally.➫Use another razor.➫Immerse the block in very cool water beforecutting.➫Change the blade pitch.

➫Decrease the section speed.➫Embed again.


Tissue Preparation

46

� Wax pieces are stuck on the razor edge.

2.4.1.3.5 SECTIONS ARE TORN

� Tissue fragment has not been well dehy-drated.

� Tissue fragment is soft.

� Block is opalescent, which is linked to thefact that water is included in the paraffin.

� Tissue is too hard.

� Wax has cooled very slowly.

2.4.1.3.6 RIBBON IS STRIATED

� Razor is scratched.

� Razor edge is dirty.

� Angle between the object and the blade istoo open.

� Sections are damaged by particles includedin the wax.

� Tissue fragment is too voluminous.

2.4.1.3.7 SECTIONS STICK TO THE BLADE

� Temperature is too high.

� Razor is scratched.

� Angle between specimen and blade is toosmall.

2.4.1.3.8 SECTIONS FLY AWAY

� There is static electricity in the air.

2.4.1.3.9 SECTIONS SHOW VIBRATIONS

� The block and/or the blade are not locked.� The block is too hard.

� Tissue is particularly calcified.

� Angle between the object and the blade istoo open.

➫Carefully clean the two sides of the razorwith a paraffin solvent.

➫Embed the piece again.

➫Embed the tissue again in paraffin with ahigher melting point.➫Embed the piece again.

➫Double embed with celloidin and paraffin.

➫Embed again with a slow cooling of paraffin.

➫Displace the razor.➫Change the razor.➫Carefully clean the two sides of the bladewith a wax solvent.➫Change the blade pitch.

➫Embed again with filtered paraffin.

➫Include with celloidin or celloidin and par-affin.

➫Immerse the block in very cool water beforecutting.➫Wait for a decrease in room temperature.➫Displace the razor.➫Change the razor.➫Change the blade pitch.

➫Increase humidity with a flame or with areceptacle full of water.

➫Lock the microtome screws.➫Reembed by melting the paraffin and embe-ding the specimen in a new wax with a lowermelting point.

➫Double embed with celloidin and paraffin.➫Reembed after decalcification.

➫Change the blade pitch.


2.4 Sections

47

2.4.2 Celloidin Sections

Blocks are cut with a special horizontal micro-tome. Sectioning it done in ethanol 70%.

➫This type of section is seldom used in classichistology.

2.4.3 Sections for Plastic Waxes

Blocks of plastic wax are placed on the stage ofa microtome with a mandrel or between twoclamps.The knife is made of glass with a special shape.A concave side is directed to the exterior, whichallows one to remove the sections easily beforeplacing them on a slide. Plastic blocks can easilybe cut automatically.

➫One can also use an ultramicrotome in semi-thin section mode.➫The sections will be 0.5 to 1 µm thick.

2.4.4 Bone Sections

Sections of wax-embedded bones are cut with aheavy microtome, which is horizontal. Sectionsare obtained with a tungsten carbide blade. ➫Sections are relatively thick: 10 µm.

2.4.5 Frozen Sections

Frozen sections are cut with a cryotome, whichis a vertical microtome that is installed in acooled room, the cryostat. The blade used isstored in this room and the temperature of theblade is that used for the section. The piece oftissue is cooled. Then it is directly embedded ina wax that is liquid at laboratory temperatureand solid at the low temperatures that are usedfor sections. This operation is done on the stageof the cryotome. Sections are obtained as with a classic microtomefor paraffin sections. Sections can be thin (5 µm).They are collected directly from the blade witha slide, where they spontaneously stick.

➫Horizontal frozen microtomes also exist.They are not installed into a cooled room. Thepiece to be cut is placed on a frozen stage, andthe blade is also cooled. The freezing systemis electric or cooled by CO2. In this case, gasis directed to the object and the blade, withoutbeing expanded.

➫Cryotomes have an antiroll plate that isplaced on the blade. This prevents the rollingof sections during preparation.


Tissue Preparation

48

2.5 ADHESION OF SECTIONS

2.5.1 Paraffin and Double-Embedded Sections

Several methods can be used to adhere paraffinand double-embedded sections.

2.5.1.1 Water adhesion

Water adhesion is the easiest method. Sectionsare arranged on the surface of a 37˚C water bath.Then they are picked up on a slide which isslipped under the section. The slides are thenwarmed at 37˚C.

➫To obtain very efficacious adhesion, it is nec-essary to remove oils from the slides by wash-ing them with ethanol and hydrochloric acid(1 vol/1 vol). Then they must be rinsed withdistilled water. It is also possible to purchasewashed and oil-free slides.

2.5.1.2 Glycerin–albumin adhesion

2.5.1.2.1 PREPARATION WITH EGG WHITE

1. Mix equal weights of egg white and glyc-erin.2. Add 0.5% sodium salicylate or thymol.3. Filter slowly.

2.5.1.2.2 MEYER’S PREPARATION

1. Add ovalbumin powder. 1 g2. Add distilled water. 100 mL3. Let dissolve.4. Add glycerin. 100 mL5. Filter.6. Add sodium salicylate (or thymol). 1 g

2.5.1.2.3 ALBUMINOUS WATER

� Distilled water 20 mL� Albuminous solution 1 mL

First protocol1. Warm albumin water in a Petri dish.

40 to 50˚C2. Place the sections on the albumin water.3. Allow sections to be perfectly plated.4. Slip the sections onto clean wet slides.5. Eliminate excess liquid.6. Dry the slides horizontally 4 h at 40˚C or vertically. 2 h at 60˚C7. Place the slides on the stage. 40 or 50˚C

➫Use a kitchen mixer to emulsify albuminmolecules.

➫This sticking agent must be preserved in arefrigerator.

➫Prepare just before use.

➫In certain cases, it is helpful to increase thesticking power by using more than 1 mL ofalbumin. In other cases, the sticking power canbe decreased by using less than 1 mL of albu-min.

➫As in the case of water sticking, it is neces-sary to use perfectly clean slides. See Section2.5.1.1.


2.5 Adhesion of Sections

49

❑ Second protocol1. Place the clean, dry slides on the stage.

40 to 50˚C2. Put some drops of albumin water on theslide.3. Place the sections on the sticking agent.4. Allow sections to be perfectly plated.5. Eliminate excess liquid.6. Dry the slides horizontally. 4 h

at 40˚C

➫As in water adhesion, it is necessary to useperfectly clean slides. See Section 2.5.1.1.

➫At minimum.

2.5.1.3 Adhesion with gelatinous water

The method is the same as the previous one, butalbumin water is replaced with a solution of 1%gelatin (warm distilled water is added until gel-atin dissolves).

➫As in water adhesion, it is necessary to useperfectly clean slides. See Section 2.5.1.1.

2.5.1.4 Adhesion on gelatinized slides

❑ Gelatinous water� Distilled water 500 mL

at 60˚C� Gelatin 2.5 g� Leave overnight. 58˚C� Add a pinch of chrome alum (fungicide).

❑ Protocol1. Dip slides in gelatinous water, two times,then let dry vertically. 2 to 3 h2. Repeat the operation.3. Store slides. 4˚C4. Stick the sections as above, replacingalbumin water with distilled water.

➫As for water adhesion, it is necessary to useperfectly clean slides. See Section 2.5.1.1.

2.5.2 Adhesion of Collodion Sections

2.5.2.1 General principles

Sticking collodion sections before staining isparticularly difficult because of embedding masselimination. In the majority of cases, sectionsare first stained by floating on dye baths. Theyare then adhered to the slides. ➫As for water adhesion, it is necessary to use

perfectly clean slides. See Section 2.5.1.1.


Tissue Preparation

50

2.5.2.2 Before staining

2.5.2.2.1 MAXIMOW’S METHOD

1. Wet section with ethanol 70%.2. Unpleat the section on the razor.3. Put the section on a glycerin–albumincoated slide.4. Blot the section with filter paper.5. Cover with oil of clove.6. Let rest. 10 min7. Immerse in ethanol 95%. 10 min8. Immerse in ethanol 100%. 2 × 10 min9. Immerse in ethanol ether (1:1).

Until celloidin dissolves10. Store in ethanol 70%.

2.5.2.2.2 CELLOIDIN ADHESION

Sections can be adhered by a solution of celloi-din in an ethanol–ether mixture (1:1).

2.5.2.2.3 GELATIN ADHESION

Sections can also be stuck with gelatin:1. Stick the sections with gelatinous waterat 1%.2. Eliminate celloidin with oil of cloves, eth-anol–ether solution and, 100% ethanol.3. Sections are ready to stain.

➫It is necessary to use perfectly clean slides.See Section 2.5.1.1.



2.5.2.3 After staining

1. Convey the sections to the stainingreagents.2. Stick sections with 2% celloidin solutionor3. Stick sections with 1% gelatinous water.4. Eliminate celloidin with oil of cloves, eth-anol–ether solution, and 100% ethanol


2.5.3 Adhesion of Plastic Wax Sections

Plastic wax sections are adhered with water byproceeding as indicated above. It is also possibleto put the sections on distilled water, then letthis water evaporate on a warming stage.



2.6 Deparaffining and hydration

51

2.5.4 Adhesion of Frozen Sections

Frozen sections are recovered directly from theblade by apposition of the slide, which has beenrefrigerated. ➫As for adhesion with water, it is necessary to

use perfectly clean slides. See Section 2.5.1.1.

2.5.5 Adhesion of Bone Sections

Sections of plastic wax-embedded bones arestained by conveying them directly into stainingbaths. It is only after this operation that sectionscan be mounted. ➫Sections are adhered to the slides with a

medium such as “Enterlan.”

2.6 DEPARAFFINING AND HYDRATION

2.6.1 Principle

Before they can be stained by dye or by ahistochemical reactive in aqueous solution,paraffin-embedded sections must be clearedand hydrated. Indeed, dyes cannot react witha tissue that is paraffin saturated. Dewaxingis done with a solvent. Then, hydration isachieved by putting slides in baths containingdecreasing concentrations of ethanol andfinally water.

2.6.2 Protocol

1. Put slides on a flame (facultative)

2. Immerse in cyclohexane. 2 × 10 min

3. Immerse in ethanol 95%. 5 min

4. Immerse in ethanol 70%. 1 min

5. Wash with tap water. 3 s

➫The flame melts the paraffin, which pro-motes the elimination of air bubbles.


Tissue Preparation

52

2.7 COLLODIONING

2.7.1 Principle

During certain histological staining or histochem-ical reactions, sections can become unstuck. Theymust be protected by a celloidin film, by placingslides with sections into a celloidin solution. Aftersolidification, celloidin will hold sections in posi-tion, even if the sticking agent fails.

2.7.2 Protocol

❑ Celloidin preparation� Celloidin 1 g� Ethanol 100% 50 mL� Ether 50 mL

❑ Collodioning1. Dewax.2. Add collodion. 1 mL3. Immerse in 95% ethanol (if albumin stick-ing) or ethanol–formalin (if gelatin sticking).4. Wash with tap water.5. Stain.

➫� Ethanol 90 mL� Formalin 10 mL

2.8 SMEARS

2.8.1 Definitions

2.8.1.1 Smears

A smear is defined as isolated cells plated on aslide.

➫“Frottis” in French and “Ausstrich” in Ger-man.➫Cells to be plated can be in a liquid mediumafter centrifugation, for example, or in a bloodsample. They can also be obtained by dilacer-ation of compact tissue.

2.8.1.2 Imprint

An imprint is done by placing a slice of an organagainst a slide or another support. The operationis done several times.

➫Empreinte in French and Abklash in Ger-man.

➫To do an imprint, the organ must not belaterally displaced during the process.


2.8 Smears

53

2.8.1.3 Squash ➫Écrasement in French and Quetschpräparatin German.

A squash is done by compressing a small frag-ment of an organ between two slides.

2.8.2 Making a Smear

2.8.2.1 Dry blood smear on slide

1. Put a drop of blood near the edge of aslide.

2. With another slide or a lamella, push thesuspension to the other side of the slide.3. Let dry.

4. Preserve the smear by immersing it in amixture of ethanol and acetic acid.

2.8.2.2 Dry blood smear on lamella

1. Put a drop of blood near the edge of aslide.2. Cover the plate with another plate.3. Let the blood stem itself.4. Separate the plates, placing a smear oneach.

➫Use perfectly clean slides. See Section2.5.1.1.

➫Drying must be immediate. To accelerate it,the technician can wave the slide in the air withfan-shaped movements.➫In certain cases, preservation can be done byother fixatives.

➫Use perfectly clean slides. See Section2.5.1.1.➫To avoid difficulties when separating theplates, hold them at a 45˚ angle.

2.8.2.3 Making a wet smear

Smears are done between a slide and a coverslip.

1. Put a drop of blood near the edge of aslide.2. With another slide or a coverslip, push thesuspension to the other side.3. Do not let dry.4. Preserve.

➫Use perfectly clean slides. See Section2.5.1.1.

➫Preserve the smear by immersing it in fixa-tive, which can be a mixture of ethanol andacetic acid.➫Preservation can also be done using osmiumtetroxide steam.


Tissue Preparation

54

2.9 CELL CULTURES

2.9.1 Monolayer Cell Culture

1. Develop a cell layer on Leighton’s tubelamella or at the bottom of a flask.2. Preserve the cell layer.3. Stain directly on the slide or on the bottomof the flask.

2.9.2 Suspension Cell Culture

A preserved block that has been obtained aftercentrifugation can be embedded in paraffin orresin, then cut and placed on a slide, like aclassic organ (Figure 2.6).

1 = Cells are obtained from a culture, asuspension, or directly from the tissue.

2 = The cell suspension is centrifuged

3A = Cells can be used to do a smear. or 3B = Treated as a compact tissue.

Figure 2.6 Treatment of cell cultures. (From Morel, G., Hybridation in Situ, Polytechnica, Editions Economica, 1998, 62. With permis-sion.)

1

2

A B

3

O


Chapter 3

Staining

0043c03 Page 55 Thursday, October 5, 2000 10:36 AM

Contents

57

Contents

3.1 Nuclear Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.2 Carmine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.1.2.1 Chemical Formula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.2.2 Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.2.3 Staining with Borated Carmine . . . . . . . . . . . . . . . . . . . . . . . . .3.1.2.4 Carmalum Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.2.5 Chromosome Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.1.3 Hematoxylin Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.3.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.3.2 Formula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.3.3 Different Staining Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.3.4 Preparative Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.1.4 Azoic Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.4.1 Azan Staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.4.2 Preparation of G and B Azocarmine . . . . . . . . . . . . . . . . . . . . .3.1.4.3 Heidenhain’s Azan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.4.4 Romeis’s Azan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.4.5 Modified Azan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.1.5 Other Staining Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.5.1 Nuclear Fast Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.5.2 Modified Azan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.1.6 Staining of Semi–thin Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.6.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.6.2 Staining with Toluidine Blue . . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.6.3 Toluidine Blue–PAS Staining . . . . . . . . . . . . . . . . . . . . . . . . . .3.1.6.4 Staining with Paraphenylenediamine. . . . . . . . . . . . . . . . . . . . .

3.2 Methods Used to Increase Contrast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.2.1 General Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.2.2 Protocols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.2.2.1 Hematoxylin–Eosin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.2.2.2 Hematoxylin–Phloxin Saffron . . . . . . . . . . . . . . . . . . . . . . . . . .3.2.2.3 Hematoxylin Picro Indigo Carmine . . . . . . . . . . . . . . . . . . . . . .3.2.2.4 Masson’s Trichroma (First Variant) . . . . . . . . . . . . . . . . . . . . . .3.2.2.5 Masson’s Trichroma (Second Variant) . . . . . . . . . . . . . . . . . . . .3.2.2.6 Masson–Goldner’s Trichroma . . . . . . . . . . . . . . . . . . . . . . . . . .3.2.2.7 Prenant Triple Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.2.2.8 Prenant Triple Staining (Method of Gabe). . . . . . . . . . . . . . . . .3.2.2.9 Ramon y Cajal’s Trichroma . . . . . . . . . . . . . . . . . . . . . . . . . . . .

595959595959606061616162626363636364656565656666666767686868686969707172727374


Staining

58

3.2.2.10 One-Time Trichroma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.2.2.11 Cleveland and Wolfe’s Trichroma . . . . . . . . . . . . . . . . . . . . . .3.2.2.12 Herlant’s Tetrachroma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.2.2.13 Paraldehyde Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3.2.2.14 Pappenheim Panoptic Staining. . . . . . . . . . . . . . . . . . . . . . . . .3.2.2.15 May–Grünwald Giemsa for Smears. . . . . . . . . . . . . . . . . . . . .

3.2.3 Preparation of Dyes Accompanying Nuclear Dyes . . . . . . . . . . . . . . .

74757676787879


3.1 Nuclear Dyes

59

3.1 NUCLEAR DYES

3.1.1 Principle

Nuclear staining uses classic histological dyesthat have been used by histologists for manyyears. These dyes have an animal or vegetalorigin, and they can be obtained by organic syn-thesis. They are not used as histochemicalreagents because their mode of action is notknown and so cannot be controlled. Their use isessentially of descriptive interest. These dyescan be used in a progressive or regressive mode.

3.1.2 Carmine

Carmine (Figure 3.1) was the first dye used inhistology. Today it is little used. It is the only dyewith an animal origin. The staining molecule iscarminic acid, which is characterized by quinonicgroups (they are chromophoric groups).

➫

Carmine is obtained by treating dry bodyextracts of female cochineal, a homopterousMexican insect, with iron alum.

3.1.2.1 Chemical formula

Figure 3.1 Carmine.

3.1.2.2 Preparation

�

Borated carmine

�

Carmalum

�

Acetocarmine

➫

See


➫

See


➫

See


3.1.2.3 Staining with borated carmine

➫

This staining method is performed on ablock before sectioning.

HO

COOH OH

OH

CO(CHOH)4CH3

CH3 O

O


Staining

60

❑

Fixative

The staining will be done on the blocks beforesectioning. Recommended fixatives containmercuric chloride (sublimate) and they aretreated for 3 days with 70% ethanol.

❑

Reagents

�

Borated carmine

�

Hydrochloride ethanol—Ethanol 70%

100 mL

—Hydrochloride acid

0.30 mL

❑

Protocol

1. Immerse in borated carmine.

3 days

2. Immerse in hydrochloride ethanol.

3 days

3. Immerse in ethanol 70%.

2 days

4. Dehydrate.5. Mount

in toto

or embed.

❑

Results

Cell nuclei are red stained.

➫

Hydrochloride ethanol is used for differen-tiation. It must be used to the point that theexcess red dye is eliminated.

➫

After staining, the piece can be encased inparaffin or celloidin. It is then sectioned andslices are mounted between a slide and a cov-erslip after dewaxing, similar to a classic sec-tion.

3.1.2.4 Carmalum staining

❑

Fixatives

Recommended fixatives contain mercuric chlo-ride (sublimate) and they are treated for 3 dayswith 70% ethanol.

❑

Reagents

�

Carmalum

�

Potassium alum 1%

❑

Protocol

1. Immerse in carmalum.

2 days

2. Immerse in potassium alum.

1 day

3. Immerse in running tap water.

1 day

4. Mount

in toto

or embed.

❑

Results

Cell nuclei are red stained.

➫

This staining method is carried out on blocksbefore sectioning.

➫

After staining, the piece can be encased inparaffin or celloidin. It is then sectioned andslices are mounted between a slide and a cov-erslip after dewaxing, similar to a classic sec-tion.

See

Section 3.1.2.3.

3.1.2.5 Chromosome staining

❑

Fixative

Use a karyotype preparation.

❑

Reagents

�

Acetocarmine


3.1 Nuclear Dyes

61

❑

Protocol:

1. Add acetocarmine.

A few drops

2. Cover with a coverslip.3. Push slightly.4. Dry with paper filter.5. Seal.

❑

Results

Chromosomes are red stained.

➫

Chromosomes can be violet stained by add-ing ferric chloride to the acetocarmine solu-tion.

3.1.3 Hematoxylin Staining

3.1.3.1 Principle

Hematoxylin is a dye that is extracted from

Hae-matoxylus campechianus

, a South Americantree. Hematoxylin is not a dye by itself. It ishematein, an oxidation product, with a quinonicgroup, that possesses chromophoric qualities.Therefore, before it is used, hematoxylin mustbe oxidized. This oxidation can be done in sev-eral ways. The classic manner is slow and con-sists of letting the hematoxylin solution beoxidized in air for 6 to 8 months. The othermethods employ an oxidant.Hematein, which does not possess an auxo-chrome group, has no affinity for tissues. It isnecessary to use a mordant, generally an alumi-num salt (iron and ammonium alum, potassiumalum, etc.). Mordants can act on the tissue beforehematoxylin action. They can also act directlyduring the hematoxylin action. The hemateinlacs that are obtained are called hemalum.Stains that are obtained can be preserved for along time without alteration, but the stain can beaffected by the action of certain acidic molecules.

➫

A lac is obtained by mordant action on thedye.

➫

Often, the terms

hemalum

and

hematoxylin

are used interchangeably.

3.1.3.2 Formula

Figure 3.2 Hematein.HO

OH

OH

O

OHO


Staining

62

Figure 3.3 Hematoxylin.

3.1.3.3 Different staining types

3.1.3.3.1

S

TAINING

PRECEDED

BY

THE

ACTION

OF

A

MORDANT

�

Regaud hematoxylin

�

Heidenhain hematoxylin

�

Mallory phosphotungstic hematoxylin

3.1.3.3.2

H

EMALUMS

These dyes are lacs obtained by mixinghemalum and hematein. They are prepared bymeans of potassium alum.

�

Hansen’s hemalum

�

Masson’s hemalum

�

Mayer’s hemalum

�

Harris’s hemalum

�

Ehrlich’s hemalum

�

Delafield’s hematoxylin

3.1.3.3.3

P

ROGRESSIVE

IRON

HEMATOXYLIN

LACS

�

Wegert’s hematoxylin

�

Iron hematoxylin

�

Groat’s hematoxylin

3.1.3.3.4

O

THER

HEMATOXYLIN

STAINS

�

Progressive chromic hematoxylin lacs

�

Hansen dihematein

�

Mallory phosphotungstic hematoxylin

➫

These staining methods can be used for sec-tions, smears, or cell cultures. Techniques arelong and difficult.

➫

Nuclei are uniformly dark stained with fewchromatin details.

➫

Methods using these dyes are the most clas-sic. They are often used with a regressive modeof staining.

➫

Groat’s hematoxylin permits a very detailedview of nuclei with chromatin details.

3.1.3.4 Preparative protocols

�

Masson’s hemalum

�

Hematoxylin

�

Groat’s hematoxylin

➫

See


➫

See


➫

See


HO

OH

OH

OH

OHO


3.1 Nuclear Dyes

63

�

Regaud’s hematoxylin

➫

See


3.1.4 Azoic Dyes

3.1.4.1 Azan staining

Azocarmine and aniline are the bases of thesetypes of staining. They allow several histologicalstructures, particularly the chromatin repartition,to be visualized with great precision.

3.1.4.2 Preparation of G and B azocarmine

➫

See


3.1.4.3 Heidenhain’s azan

❑

FixativeAll fixatives can be used, although fixatives withchromium or osmium should be avoided.❑ Reagents

� G or B azocarmine� Diluted Heidenhain blue� Aniline 1% in 70% ethanol� Acetic acid 1% in 95% ethanol� Phosphotungstic acid 5% in distilled water

❑ Protocol1. Dewax; collodion, if necessary; hydrate.2. If sections have been provided from tissuepreserved with a fixative containing picricacid, the picric acid can be eliminated by 30min in ethanol/aniline mixture.3. Incubate in G azocarmine. 1 h

at 60˚Cor3. Incubate in B azocarmine. 1 h

at RT4. Rinse in distilled water.5. Differentiate by aniline ethanol until analmost pure nuclear staining is obtained.

6. Immerse in acetic ethanol. 30 s7. Wash with distilled water.8. Immerse in phosphotungstic acid.30 min9. Wash with distilled water.10. Immerse in Heidenhain blue. 1 h11. Differentiate blue with 95% ethanol.

➫The stage of picric acid elimination isoptional.

➫Differentiation must be performed undermicroscope control. Caution: Differentiationcan be extremely fast and can provoke theelimination of nuclear staining. In this case,the only thing to do is restain!➫Acetic ethanol stops azocarmine differenti-ation. Stay of sections can be prolonged.➫Phosphotungstic acid acts as a mordant andmakes Heidenhain blue staining possible. Italso prolongs azocarmine differentiation.


Staining

64

12. Dehydrate directly with 100% ethanol.13. Mount.

❑ ResultsNuclei and certain cytoplasms are red stained;other cytoplasms are yellow or gray. Collagenis blue stained. Secretions can be differentdepending on their nature. Acid mucopolysac-charides are blue stained.

➫Nuclei are very well stained. If the protocolis well done, all details of chromatin can beobserved.

3.1.4.4 Romeis’s azan

❑ FixativeAll fixatives can be used, although fixatives withchromium or osmium should be avoided.❑ Reagents

� G or B azocarmine� Aniline blue� Aniline 1% in 70% ethanol� Acetic acid 1% in 95% ethanol

❑ Protocol1. Dewax; collodion, if necessary; hydrate.2. If sections have been provided from tissuepreserved with a fixative containing picricacid, the picric acid can be eliminated by 30min in an ethanol/aniline mixture.3. Incubate in G azocarmine. 1 h


at RT4. Rinse in distilled water.5. Differentiate by aniline ethanol until analmost pure nuclear staining is obtained.

6. Immerse in acetic ethanol. 30 s7. Rinse in distilled water. 30 s8. Immerse in phosphomolybdic G orange.5 min9. Wash with distilled water.10. Immerse in aniline blue. 10 min11. Differentiate blue by 95% ethanol.12. Dehydrate.13. Mount.

❑ ResultsNuclei and certain cytoplasms are red stained, othercytoplasms are yellow or gray. Collagen is blue stained.Secretions can be different depending on their nature.Acid mucopolysaccharides are blue stained.

➫The stage of picric acid elimination isoptional.

➫Differentiation must be performed undermicroscope control. Caution: Differentia-tion can be extremely fast and can pro-voke the elimination of nuclear staining.In this case, the only thing to do is restain!➫Acetic ethanol stops azocarmine differenti-ation. Stay of sections can be prolonged.

➫Nuclei are well stained with Heidenhain’sazan. If the protocol is well done, all the detailsof chromatin can be observed.


3.1 Nuclear Dyes

65

3.1.4.5 Modified azan

Nuclei are stained by nuclear fast red. ➫This technique is easy to perform success-fully. But details of nuclei are not as observableas with a true azan (Romeis or Heidenhain).➫See Section (3.1.5.2).

3.1.5 Other Staining Techniques

3.1.5.1 Nuclear fast red

3.1.5.1.1 DEFINITION

Nuclear fast red is a synthetic dye with ananthraquinonic nature. It can be used alone orin combination with other dyes.

3.1.5.1.2 FORMULA

See Figure 3.4.

3.1.5.1.3 STAINING

❑ Reagent� Nuclear fast red

❑ Protocol1. Dewax.2. Hydrate.3. Immerse in nuclear fast red. 2 min4. Rinse with distilled water.5. Dehydrate, mount.

❑ ResultsNuclei are red stained.

Figure 3.4 Nuclear fast red.

➫See Chapter 7: Preparation of Products.

➫This method is useful for obtaining a quickstaining when another tissue component isvisualized with a dark color.

3.1.5.2 Modified azan

Nuclei are stained by nuclear fast red.❑ Reagents

� Nuclear fast red� Aniline blue� Molybdic G orange

➫See Chapter 7: Preparation of Products.➫See Chapter 7: Preparation of Products.➫See Chapter 7: Preparation of Products.

O

O


Staining

66

❑ Protocol1. Dewax, hydrate.2. Immerse in nuclear fast red. 15 min3. Rinse.4. Immerse in molybdic G orange. 5 min5. Rinse with water.6. Immerse in aniline blue. 2 to 5 min7. Wash with distilled water.8. Immerse in 95% ethanol.9. Dehydrate, mount.

❑ ResultsNuclei appear similar to those stained byHeidenhain’s or Romeis’s azan.

➫Washing with distilled water eliminatesexcess aniline blue.

3.1.6 Staining of Semi-thin Sections

3.1.6.1 Principle

Semi-thin sections are particularly used to control thetissue present before sectioning it into ultra-thin sec-tions for observation with electron microscopes.Semi-thin sections can be stained by most stainingtechniques that are used in classic histology. For stain-ing, proceed as indicated in the first part of this chap-ter, beginning with dewaxing and hydration.Mounting can be done with Eukitt or similar medium.In addition to classic staining, several specific andquick techniques allowing examination also exist.

3.1.6.2 Staining with toluidine blue

❑ FixativeAll classic fixatives can be used. Glutaraldehyde–paraformaldehyde solution is commonly used.❑ Reagents

� Toluidine blue 0.5 g� Sodium carbonate (0.25g/L) 100 mL

pH 11❑ Protocol

1. Put the slide with sections on a plate 80˚C2. Add a drop of filtered dye.3. Let evaporate.4. Rinse with distilled water.5. Let evaporate.

❑ ResultsTissues stain different shades of blue. Nucleicacids are purple colored.

➫Purple staining of nucleic acids and other sub-stances is linked to the metachromatic qualitiesof the dye.


3.1 Nuclear Dyes

67

➫This staining method yields several colorswith only one dye. It is useful for obtainingquick results.

3.1.6.3 Toluidine blue–PAS staining

❑ FixativeAll classic fixatives can be used. Glutaraldehyde–paraformaldehyde solution is commonly used.❑ Reagents

� Periodic acid 1%� Schiff reagent� Sulfurous water—Sodium metabisulfite 1 mL—Distilled water 20 mL—Hydrochloric acid M 1 mL� Toluidine blue (pH 11)—Toluidine blue 2.5 g—Sodium carbonate 0.5 g/L 50 mL—Stir, let boil, filter.

❑ ProtocolSections can be stained before mounting on theslides by carrying them to the different dye ves-sels with forceps. Sections can also be mountedon a slide before staining. In this case, use theclassic method:

1. Immerse in periodic acid. 15 min2. Rinse with distilled water. 2 × 2 min3. Immerse in Schiff reagent. 30 min4. Immerse in sulfurous water. 2 × 2 min5. Rinse with distilled water.6. Immerse in toluidine blue. 1 min7. Rinse with distilled water.8. Mount sections on a slide if staining hasbeen done on sections.9. Eliminate water with filter paper.10. Let dry in air.

❑ ResultsGlycogen is pink; cytoplasm and nuclei are blueto purple.

➫Purple staining of nucleic acids and othersubstances is linked to the metachromatic qual-ities of the dye. This staining is useful forvisualizing nucleic acids with sugars and othertissue components.

3.1.6.4 Staining with paraphenylenediamine

❑ FixativeAll classic fixatives can be used. Glutaraldehyde–paraformaldehyde solution is commonly used.


Staining

68

❑ Reagents� Paraphenylenediamine—Paraphenyldiamine 1 g—Ethanol 95% 100 mL

❑ Protocol1. Put slides with sections on a plate. 80˚C2. Let evaporate.3. Rinse with distilled water.

❑ ResultsStructures are pink stained, as are nuclei. ➫This staining method yields quick results.

3.2 METHODS USED TO INCREASE CONTRAST


Coloration must permit immediate identificationof the tissue; well-contrasted staining makes thiseasy. Further, the coloration must permit goodhistological pictures.Generally, a nuclear dye and a cytoplasmic dyeare used. It is then possible to add staining ofcollagen or elastic fibers. Secretions can also bevisualized and histochemical methods will per-mit determination of their chemical nature.

The list that follows is not exhaustive. Referenceto more-specialized books will permit one tomodify each staining method or to find moreunusual techniques.

➫For most stainings methods, one dye can bereplaced with another that possesses closelyrelated properties. For example, hematoxylincan be replaced with nuclear fast red.

3.2.2 Protocols

3.2.2.1 Hematoxylin–eosin

This staining method uses a nuclear dye(hemalum or Groat’s hematoxylin) and a cyto-plasmic dye (eosin or phloxin). ❑ FixativeAll fixative agents are convenient.❑ Reagents

� Groat’s hematoxylin� Eosin 1% or phloxin 1%

➫See Chapter 7: Preparation of Products.➫See Chapter 7: Preparation of Products.


3.2 Methods Used to Increase Contrast

69

❑ Protocol1. Dewax, hydrate.2. Immerse in Groat’s hematoxylin. 5 min3. Wash with tap water to obtain a bluehematoxylin.4. Immerse in eosin or phloxin. 30 s5. Wash with tap water.6. Dehydrate, mount.

❑ ResultsNuclei are stained dark blue (they are brown ifthe hematoxylin is too old); acidophilic cyto-plasm is pinkish. Certain secretions remainuncolored.

➫Rinsing duration after adding Groat’s hema-toxylin is equal to the dye bath duration. WhenGroat’s hematoxylin becomes too old, its stain-ing qualities are weakened; nuclei are brown-ish colored, not dark blue, and it is necessaryto use a new dye solution. However, to prolongthe staining qualities of hematoxylin, it is pos-sible to prolong the bath duration to 10 or 15min and rinse for the same amount of time.➫This staining method yields quick results,but it is not sufficiently selective to visualizethe tissue components very precisely. But it isvery often used.

3.2.2.2 Hematoxylin–phloxin saffron

This staining method uses hemalum (or hema-toxylin) as a nuclear dye and phloxin as a cyto-plasmic dye. Saffron is collagen specific.❑ FixativeAll classic fixative agents are convenient.❑ Reagent

� Hemalum or Groat’s hematoxylin� Phloxin 0.5% or 1% in distilled water� Saffron obtained by distillation in ethanol

❑ Protocol1. Dewax, hydrate.2. Immerse in Groat’s hematoxylin. 5 min3. Wash with tap water. 5 min4. Immerse in phloxin. 3 min5. Rinse.6. Immerse in 95% ethanol. 2 min7. Immerse in 100% ethanol. 2 min8. Immerse in saffron. 10 min9. Immerse in 100% ethanol. Quickly10. Immerse in butanol, cyclohexane.11. Mount.

❑ ResultsNuclei are blue stained; cytoplasm, muscle fibers,and red blood cells are red; and collagen is yellow.

➫This is used as a standard staining methodin pathologic anatomy. It comes from Mas-son’s trichroma.


3.2.2.3 Hematoxylin picro indigo carmine

This staining method uses hemalum or Groat’s


Staining

70

hematoxylin as a nuclear dye and a mix of dyes,picro indigo carmine, as acytoplasmic dye.❑ Fixative

� Masson’s hematoxylin

� Calleja’s picro indigo carmine❑ Protocol

1. Dewax, hydrate.2. Immerse in hematoxylin. 3 min3. Wash with tap water to obtain a darkbrown staining.4. Immerse in picro indigo carmine. 30 s5. Immerse in 100% ethanol. 10 min6. Dehydrate.7. Mount.

❑ ResultsNuclei and basophilic cytoplasm are brown col-ored. Acidophilic cytoplasm and nucleoli areyellow or green stained. Collagen fibers areblue; red blood cells are yellow. Glycoproteinsare brown. Secretions are yellow or greenstained.

3.2.2.4 Masson’s trichroma (first variant)

➫Masson’s hematoxylin can be replaced withGroat’s hematoxylin. See Chapter 7: Prepara-tion of Products.➫See Chapter 7: Preparation of Products.

➫This staining method is useful for obtainingvery quick results for a morphological appre-ciation.

➫Variations of stain can be observed on thesections.

The nuclear dye can be Masson’s hematoxylinor one of several hematoxylin solutions. Thecytoplasmic dye is fuchsin. An aniline-deriveddye allows differentiation of collagen fibers.❑ FixativeAll fixative agents are convenient.❑ Reagent

� Regaud’s hematoxylin� Acidic fuchsin culvert� Fast green� Iron and ammonium alum 5%� Picric acid saturated in ethanol� Acetic water 0.5%� Phosphomolybdic acid 1%

❑ Protocol1. Dewax, hydrate.2. Immerse in iron and ammonium 15 min alum. at 50˚C3. Immerse in Regaud’s 15 minhematoxylin. at 50˚C4. Differentiate by picric ethanol to obtaingray reflection on the sections.5. Wash with tap water. 10 min

➫This technique has numerous variants(hematoxylin, phloxin, and saffron, or evenMasson–Goldner’s trichroma).




71

6. Immerse in acid fuchsin culvert. 1 minat 50˚C

7. Differentiate with phosphomolybdic acid.5 to 10 min

at 50˚C

8. Rinse with acetic acid.9. Immerse in fast green. 10 min10. Rinse with acetic water.11. Dehydrate, mount.

❑ ResultsNuclei are brownish blue; cytoplasm is red;secretions are red or green; muscles and collagenfibers are green stained.

➫Monitor differentiation duration carefully. Ifphosphomolybdic acid action is prolonged, allthe nuclear dye can be extracted from the sec-tion. Conversely, a fast differentiation can leadto an excess of dye. The entire section is “clog-ged” by hematoxylin; nuclei appear to be uni-formly dark and chromatin cannot be seen.

➫This staining method yields good results formorphological studies because the differentparts of tissue are very concentrated.

3.2.2.5 Masson’s trichroma (second variant)

The nuclear dye can be Masson’s hematoxylinor one of several hematoxylin solutions. Thecytoplasmic dye is fuchsin. An aniline-deriveddye allows differentiation of collagen fibers.❑ Fixative All fixatives are convenient.❑ Reagent

� Regaud’s hematoxylin� Aniline blue saturated in acetic water 2.5%� Acidic fuchsin� Iron and ammonium alum 5%� Phosphomolybidic acid 1%� Acetic water 1%� Acetic ethanol (100%) 1%

❑ Protocol1. Dewax, hydrate.2. Immerse in iron and ammonium alum. 24 h3. Immerse in Regaud’s hematoxylin.30 min

at 50˚C4. Wash with distilled water.5. Let sections drain.6. Differentiate by iron and ammonium alum(or by picric acid saturated in ethanol 95%)to obtain a pure nuclear staining (darkbrown).7. Wash with distilled water.8. Immerse in acidic fuchsin. 5 min9. Rinse with distilled water.10. Differentiate with phosphomolybdicacid. 5 min


➫Do not wash sections between phosphomo-lybdic acid and aniline blue.


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72

11. Immerse in aniline blue. 5 min12. Differentiate in acetic water. 5 to 30 min13. Dip in acetic ethanol 100%. Passage14. Dehydrate, mount.

❑ ResultsNuclei are black; cytoplasm is red; muscles, col-lagen fibers, and mucus are blue stained.

➫A passage consists of quickly dipping a slideinto the mixture. An increased stay can havethe same effect as a prolonged differentiation.

➫This method yields good results for morpho-logical studies. However, the blue staining ofconnective tissues provides a less-contrastedpicture of the sections than does the first variant.

3.2.2.6 Masson–Goldner’s trichroma

The principle is the same as with Masson’strichroma. The nuclear dye is Groat’s hematoxylin.❑ FixativeAvoid fixative with osmium tetroxide❑ Reagents

� Groat’s hematoxylin

� Fuchsin culvert� Molybdic G orange� Acetic sulfo green� Acetic water 1%

❑ Protocol1. Dewax, hydrate.2. Immerse in Groat’s hematoxylin. 5 min3. Add tap water to obtain a blue staining ofsections.4. Immerse in fuchsin culvert. 5 min5. Rinse with acetic water.6. Immerse in molybdic G orange. Passage7. Immerse in acetic sulfo green. 10 min8. Rinse with acetic acid.9. Dehydrate.10. Mount.

❑ ResultsNuclei are black or dark blue. The bottom ofcells is gray; acidophilic cytoplasm is pink;secretions are red or green stained. Muscles arered, and collagen fibers green.

➫Groat’s hematoxylin gives a particularly pre-cise staining to nuclei. It is possible to see allthe details of chromatin repartition. See Chap-ter 7: Preparation of Products.➫See Chapter 7: Preparation of Products.➫See Chapter 7: Preparation of Products.➫See Chapter 7: Preparation of Products.

➫Groat’s hematoxylin must be exclusivelyused.

➫It is often necessary to change acetic waterafter each slide passage.

➫This trichrome gives very good results formorphological studies. Nuclei are verydetailed, and the different parts of tissues arewell visualized.

3.2.2.7 Prenant triple staining

This staining technique is derived from the oldmethod called “iron hematoxylin.” Its results are



73

dependent upon preservation and the stainingprotocol.

❑ FixativeAvoid fixatives with osmium tetroxide.❑ Reagent

� Eosin 1%� Regaud’s hematoxylin� Aqueous solution of sulfo green 0. 5%� Iron and ammonium alum 0.5%

❑ Protocol1. Dewax, hydrate.2. Immerse in eosin. 10 min3. Immerse in iron and ammonium alum.14h4. Immerse in Regaud’s hematoxylin. 24 h5. Differentiate with iron and ammonium alum.6. Immerse in sulfo green. 20 to 30 s7. Dehydrate by ethanol 100%.8. Mount.

❑ ResultsNuclei and basophilic cytoplasm are dark brownstained; collagen is green. Cytoplasm, nucleoli,and erythrophilic secretions are green, as arecytoplasm and cyanophilic secretions.


➫Stop the differentiation when the generalcoloration of the slide is gray.

➫This can be a good method for a morpho-logical study. However, it is lengthy.

3.2.2.8 Prenant triple staining (method ofGabe)

❑ FixativeAvoid fixatives with osmium tetroxide.❑ Reagent

� Groat’s hematoxylin� Eosin sulfo green� Acetic water 0.5%

❑ Protocol1. Dewax, hydrate.2. Immerse in hematoxylin. 5 min3. Wash with tap water.4. Immerse in eosin sulfo green. 10 min5. Rinse quickly with distilled water, or holdin acetic water.6. Dehydrate.7. Mount.

❑ ResultsNuclei and basophilic cytoplasm are dark brownstained; collagen is green. Cytoplasm, nucleoli,and erythrophilic secretions are pink. Cytoplasmand cyanophilic secretions are green.


➫This is a good method for morphologicalstudies, and it is quicker than the originalmethod.


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74

3.2.2.9 Ramon y Cajal’s trichroma

❑ FixativeAll fixative agents are convenient.❑ Reagents

� Ziehl’s fuchsin diluted at 1:5 with distilledwater

� Picro indigo carmine� Acetic water 0.2%

❑ Protocol� After fixative without osmium tetroxide:1. Dewax, hydrate.2. Immerse in Ziehl’s fuchsin. 10 min3. Wash with tap water.4. Wash with acetic water to the point offuchsin excess.5. Immerse in picro indigo carmine. 5 min6. Immerse in acetic water. 10 min7. Add ethanol 100% until departure of red isstopped.8. Continue dehydration.9. Mount.� After a preservation with osmium tetroxide:1. Dewax, hydrate.2. Immerse in Ziehl’s fuchsin. 45 min

at 60˚C3. Immerse in picro indigo carmine. 30 s4. Immerse in picro indigo carmine. 5 min5. Immerse in acetic water. 10 min6. Add ethanol 100% until departure of red isstopped.7. Continue dehydration.8. Mount.

❑ ResultsNuclei and basophilic cytoplasm are red colored.Acidophilic cytoplasm is green or gray; collagenis blue. Secretions are green, blue, or red. Mucusis orange or purple colored.



➫This method provides very well-contrastedresults.

3.2.2.10 One-time trichroma

This is a quick method. Nuclei and cytoplasm arestained by azorubine. Phosphomolybdic acid isthe mordant. Solid green stains connective fibers,and naphthol yellow stains red blood cells.❑ FixativeAvoid, if possible, mixtures with dichromate orosmium tetroxide.



75

❑ Reagent� One-time trichroma

❑ Protocol➫See Chapter 7: Preparation of Products.

1. Dewax, hydrate.2. Immerse in one time trichroma. 10 min3. Rinse.4. Dehydrate, rinse.5. Mount.

❑ ResultsNuclei and cytoplasm are red stained; connec-tive fiber is green; and red blood cells are yellow.Mucopolysaccharides are more often green.

➫If the fixative contains osmium tetroxide:� One-time trichroma 45 min

at 60˚C➫Sections can be stored in acetic water 1%after rinsing.

➫If sections have an excess of red, treat themwith yellow naphthol in saturated solution.➫This method is quick, but is often unsuccess-ful and results are sometimes deceiving.

3.2.2.11 Cleveland and Wolfe’s trichroma

Cleveland and Wolfe’s technique is used to dis-tinguish different secretory cell types belongingto adenohypophysis, which are characterized bytheir tinctorial properties. The original techniquedeveloped by Cleveland, Rucker, and Wolfe(1932) is not often used today. However, Her-lant’s modified method (1956) is used for study-ing cell types, along with other techniques,particularly for immunocytochemical detectionof hormones.❑ FixativeAll classic fixatives can be used. Halmi’s liquidis recommended.❑ Reagents

� Erythrosin 1%� G orange 2% into phosphotungstic acid 1%� Aniline blue 1%

❑ Protocol1. Dewax, hydrate.2. Immerse in erythrosin. 3 min3. Rinse with distilled water.4. Immerse in phosphotungstic G orange. 30 s5. Rinse with distilled water.6. Immerse in aniline blue. 1 to 2 min7. Rinse with distilled water.8. Dehydrate.9. Mount.

❑ ResultsNuclei are blue stained, nucleoli are pinkish,somatotropic cells are pinkish, thyreotropic cellsare purple, corticotropic cells are pale purple,lactotropic cells are orange stained, and gona-dotropic cells are blue with pinkish granulations.

➫Cleveland and Wolfe’s technique is essen-tially descriptive because it does not give anyindication concerning the chemical structureof the cell content. To study the pituitary, itis important to validate the method with aprecise immunocytochemical study that willprovide precise hormonal information foreach cell category.

➫Cleveland and Wolfe’s trichroma can beused to visualize each tissue type.


➫This method is quick and provides very goodvisualizations of nuclei and the different partsof tissue. It is used for morphological studies.


Staining

76

3.2.2.12 Herlant’s tetrachroma

This method was initially used to stain the pitu-itary gland, but it can be used for all types oftissues and provides well-contrasted results.❑ FixativeThe best fixative contains mercuric chloride(Susa, Halmi). Mixtures containing potassiumdichromate are not recommended. Carnoy’sfluid also gives good results.❑ Reagents

� Erythrosin (acetic solution)� Diluted Heidenhain blue� Acidic alizarin blue� Phosphomolybdic acid 5%

❑ Protocol1. Dewax, hydrate.2. Immerse in erythrosin. 10 min3. Rinse with distilled water.4. Immerse in Heidenhain blue. 5 min5. Rinse with distilled water.6. Immerse in alizarin blue. 10 min7. Rinse with distilled water.8. Immerse in phosphomolybdic acid. 10 min9. Dehydrate.10. Mount.

❑ ResultsChromatin is blue colored; nucleoli are red; cyto-plasm and secretions are red or purple; mucus isblue; and cartilage and collagen are dark blue.


➫Erythrosin can be differentiated by ethanol70%.

➫This method is quick and provides very goodvisualization of nuclei and the different partsof tissue. It is used for morphological studies.

3.2.2.13 Paraldehyde fuchsin

This technique has been used to stain elasticfibers (Gomori, 1950). It stains most secretionsif oxidization has previously been performed.❑ FixativeAll classic fixatives are convenient, but prolongedpreservation by dichromate should be avoided.

FIRST VARIANT: WITHOUT OXIDIZATION

❑ Reagents� Paraldehyde fuchsin� Groat’s hematoxylin� Picro indigo carmine

� Hydrochloric acid 0.5% in ethanol 100%

➫See Chapter 7: Preparation of Products.➫Groat’s hematoxylin can be replaced withnuclear fast red; nuclei will be red stained. SeeChapter 7: Preparation of Products.➫See Chapter 7: Preparation of Products.



77

❑ Protocol1. Dewax, hydrate.2. Immerse in paraldehyde fuchsin. 5 min3. Wash in tap water.4. Dehydrate with ethanol 100%.5. Differentiate with hydrochloride ethanoluntil dye stops running off.6. Wash with tap water.7. Immerse in Groat’s hematoxylin. 5 min8. Wash with tap water.9. Immerse in picro indigo carmine. 30 s10. Dehydrate.11. Mount.

❑ ResultsNuclei are dark brown, cytoplasm are green stained,elastic fibers are purple, and collagen fibers are blue.

SECOND VARIANT: WITH OXIDIZATION

❑ Reagents� Paraldehyde fuchsin� Groat’s hematoxylin� Picro indigo carmine� Gomori oxidizing—KMnO4 2.5% 15 mL—H2SO4 5% 15 mL—Distilled water 90 mL� Aqueous solution of sodium bisulfite or

metabisulfite❑ Protocol

1. Dewax, hydrate.2. Immerse in Gomori oxidizer. 20 to 30 s3. Rinse with distilled water.4. Whiten sections by a quick treatment withsodium bisulfite.5. Rinse with tap water. 5 min6. Immerse in paraldehyde fuchsin. 2 min7. Wash with tap water. 2 min8. Immerse in Groat’s hematoxylin. 5 min9. Wash with tap water. 5 min10. Immerse in picro indigo carmine. 30 s11. Dehydrate.12. Mount.

❑ ResultsElastic fibers and certain secretions are purplestained, nuclei are brown-black, cytoplasm isgreen, and collagen fibers are blue stained.

➫This method is often used to stain elasticfibers on a tissue.


➫This method is often used to stain elasticfibers and secretions on a tissue.


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78

3.2.2.14 Pappenheim panoptic staining

This staining method, first used for smears, isalso used for sections. It allows one to visualizehematopoietic organs.

❑ FixativeAll the classic fixatives are convenient.❑ Reagents

� May–Grünwald—May–Grünwald 10 mL—Distilled water 80 mL� Giemsa—Giemsa 10 mL—Distilled water 750 mL� Acetic water 0.15%

❑ Protocol1. Dewax, hydrate.2. Immerse in May–Grünwald. 20 min

at 35˚C3. Rinse.4. Immerse in Giemsa. 40 min

at 35˚C5. Differentiate with acetic water.6. Wash with tap water.7. Dehydrate by acetone.8. Mount.

❑ ResultsNuclei are dark purple, basophilic cytoplasm isblue, acidophilic cytoplasm is red, collagen ispale blue, mucus is blue or purple, muscles arepinkish, and cartilage is blue.In blood cells, granulations of lymphoid cellsare purple, that of myeloid cells are violet. Neu-trophilic granulations are brownish or bluish.Erythrosinophilic granulations are brick red, andbasophilic granulations are blue colored.

➫This relatively quick and easy method yieldsvery good pictures of tissue. It permits visual-ization of the different blood cells on sections.

3.2.2.15 May–Grünwald Giemsa for smears

❑ FixativeSee special preservation of smears.❑ Reagents

� May–Grünwald� PBS pH 6.7—Crystallized disodium phosphate (35.8g/L) 433 mL—Monosodium phosphate (13.805 g/L)

567 mL



79

� Giemsa—Staining solution 3 drops—PBS 2 mL

❑ Protocol1. Immerse in May–Grünwald. 3 min

➫If crystallized disodium phosphate is notavailable, the following may be used:

2. Immerse in PBS. 3 min3. Immerse in Giemsa. 20 min4. Rinse with water.5. Let dry.6. Mount with a hydrophobic wax.

❑ ResultsIn the blood cells, granulations of lymphoid cellsare purple, those of myeloid cells are violet.Neutrophilic granulations are brownish or blu-ish. Erythrosinophilic granulations are brick redand basophilic granulations are blue colored.

� Anhydrous phosphate 14.8 g� Distilled water 1000 mL

➫This relatively quick and easy method per-mits visualization of the different blood cellson sections.

3.2.3 Preparation of Dyes Accompanying Nuclear Dyes

� Alizarin acid blue ➫See Chapter 7: Preparation of Products.

� Aniline blue ➫See Chapter 7: Preparation of Products.

� Heidenhain blue ➫See Chapter 7: Preparation of Products.

� Eosin ➫See Chapter 7: Preparation of Products.

� Eosin–light green ➫See Chapter 7: Preparation of Products.

� Erythrosin ➫See Chapter 7: Preparation of Products.

� Erythrosin–G orange ➫See Chapter 7: Preparation of Products.

� Fast green ➫See Chapter 7: Preparation of Products.

� Acidic fuchsin ➫See Chapter 7: Preparation of Products.

� Acidic fuchsin and culvert ➫See Chapter 7: Preparation of Products.

� Altmann’s fuchsin ➫See Chapter 7: Preparation of Products.

� Paraldehyde fuchsin (Gabe’s formula) ➫See Chapter 7: Preparation of Products.

� Ziehl’s fuchsin ➫See Chapter 7: Preparation of Products.

� Phloxin ➫See Chapter 7: Preparation of Products.

� Calleja’s picro indigo carmine ➫See Chapter 7: Preparation of Products.


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80

� Saffron ➫See Chapter 7: Preparation of Products.

� One-time trichroma (Gabe’s formula) ➫See Chapter 7: Preparation of Products.

� One-time trichroma (Martoja’s formula) ➫See Chapter 7: Preparation of Products.

� Acetic light green ➫See Chapter 7: Preparation of Products.


Chapter 4

HistochemicalMethods

0043c04.fm Page 81 Thursday, October 5, 2000 10:37 AM

Contents

83

Contents

4.1 General Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.1.1 General Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.1.2 Detection of Puric and Pyrimidic Bases . . . . . . . . . . . . . . . . . . . . . . .

4.1.2.1 Caspersson’s Spectrophotometric Method. . . . . . . . . . . . . . . . .4.1.2.2 Danielli’s Tetrazoreaction after Benzoylation or Acetylation . .

4.1.3 Pentose Visualization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.1.3.1 Method of Turchini et al. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.1.3.2 Other Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.1.4 Visualization of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.1.4.1 Hartig–Zacharias’s Method . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.1.4.2 Danielli’s Tetrazoreaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.1.4.3 T chloramine–Schiff Method . . . . . . . . . . . . . . . . . . . . . . . . . . .4.1.4.4 Visualization of Proteins by Coomassie Blue . . . . . . . . . . . . . .

4.2 Basophilic Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.1 General Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.2 Gallocyanin Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.2.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.2.2 Chemical Formula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.2.3 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.3 Methyl Green Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.3.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.3.2 Chemical Formula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.3.3 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.4 Pyronine Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.4.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.4.2 Chemical Formula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.4.3 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.5 Pappenheim–Unna Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.5.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.5.2 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2.6 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.6.1 Ribonuclease Brachet’s Test . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.6.2 RNA Extraction by Hydrochloric Acid . . . . . . . . . . . . . . . . . . .

4.2.7 Mann–Dominici’s Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.8 Semi-thin Section Staining with Toluidine Blue . . . . . . . . . . . . . . . . .4.2.9 Love and Liles’s and Love and Suskind’s Methods . . . . . . . . . . . . . . .

4.2.9.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.9.2 Methods for Paraffin Sections . . . . . . . . . . . . . . . . . . . . . . . . . .4.2.9.3 Method for Smears . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.3 Feulgen and Rossenbeck Nuclear Reaction . . . . . . . . . . . . . . . . . . . . . . . . . .4.3.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.3.1.1 General Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.3.1.2 Chemical Reaction of Acidic Hydrolysis. . . . . . . . . . . . . . . . . .4.3.1.3 Schiff Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.3.2 Schiff’s Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8585858585888889909090909192929292929293939393949494949595959696969798989898

100101101101102102102


Histochemical Methods

84

4.3.3 Histochemical Practice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.3.3.1 Preservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.3.3.2 Hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.3.4 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.3.4.1 General Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.3.4.2 First Variant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.3.4.3 Second Variant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.3.5 Alternative Methods to Feulgen’s Reaction . . . . . . . . . . . . . . . . . . . . .4.3.5.1 General Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.3.5.2 Thionin–SO

2

Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.3.5.3 Himes and Moriber’s Method . . . . . . . . . . . . . . . . . . . . . . . . . .4.3.5.4 Benson’s Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.3.5.5 Silver Methenamine Method . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.4 Other Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.4.1 General Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.4.2 Periodic Acid and Silver Diamine Method . . . . . . . . . . . . . . . . . . . . .

4.4.2.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.4.2.2 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.4.3 Radioactive Actinomycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.4.4 Quantification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

103103104105105105106107107107108108109110110110110111111111


4.1 General Methods

85

4.1 GENERAL METHODS


Nucleic acids are linked with proteins to formnucleoproteins. The fundamental unit is alwaysa nucleotide composed of a phosphoric acidmolecule, a pentose, and a puric or pyrimidicbase. Histochemical methods permitting visual-ization of nucleic acids can be classified intofour categories that are functions of the visual-ized molecular component:

�

Puric and pyrimidic acids permit nucleicacid detection by use of ultraviolet spec-trography.

�

Presence of phosphoric acids gives an elec-tronegative character to nucleic acids.

�

Presence of pentose permits certain meth-ods to be used for sugar detection.

�

Presence of a proteinic part permits meth-ods for protein detection to be used.

4.1.2 Detection of Puric and Pyrimidic Bases

4.1.2.1 Caspersson’s spectrophotometricmethod

Puric and pyrimidic bases are the only tissuemolecules that strongly absorb ultraviolet wave-length at 260 nm. This method can be used onsmears, cell cultures, sections, irrespective of thefixative used.RNA and DNA absorption curves are similar.Therefore, this method may only be used fornormal sections and for sections from which oneor the other nucleic acid has been extracted.

➫

This spectrophotometric method is rarelyused.

4.1.2.2 Danielli’s tetrazoreaction afterbenzoylation or acetylation

4.1.2.2.1

P

RINCIPLE

Danielli’s tetrazoreaction was first used to visu-alize aromatic or heterocyclic amino acids, bymeans of stained azoic molecules (Figure 4.1).



86

The first reaction was a diazotation of aminoacids, where a diazonium salt was placed incontact with a tissue and the molecules of inter-est were visualized. However, the intensity ofthis reaction was minuscule. Danielli (1947)used a reaction based on a double coupling. Thefirst reaction consists of reacting the tissue ele-ment weakly stained with a diazonium salt,which gives a first product. Then, the product iscombined with a naphthol, and the result is anintense reaction, the color of which varies withthe salt used. This method, first used to visualizeproteins, has been adapted for nucleic acids. Fornucleic acids benzoylation or acetylation mustbe performed before staining. Chromosomes areintensely stained.

4.1.2.2.2

O

RIGINAL

METHOD

The original method consists of combining tet-razoted benzidine and certain amino acids, at analkaline pH and at a low temperature. The result-ing molecule is then coupled to an aromaticamine. It is better to replace tetrazoted benzidinewith fast blue B.

❑

Fixative

All classic fixatives are convenient.

❑

Reagent

�

Fast blue B (orthodianisidine) 0.2% inveronal buffer pH 9.2

�

H acid 2% in veronal buffer pH 9.2

➫

The salt generally used in the reactions isorthodianisidine, also called fast blue B.Another salt that is often used is H acid, whichis characterized by a

β

-naphthol function. Tis-sue and cell groups are brown stained.

➫

Certain authors have supposed that nucleicacids were visualized because they were asso-ciated with proteins. Whatever the reaction,this method is useful for visualizing nucleicacids.

Figure 4.1 Danielli’s tetrazoreaction.

➫

However, in the original method, avoidanceof mixtures with formalin is recommended.

➫

See


➫

See


SO3H

SO3HHO3S

H2O

SO3 H

NH2

NH2

OH

OH

OH

OH

NN N

OH

OHNN N N

N

HO N N OHN N R

+

R

R

+


4.1 General Methods

87

�

Veronal buffer:—Hydrochloride acid 8.35 g/L

231 mL

—Sodium veronal 20.618 g/L

769 mL

❑

Protocol

1. Dewax, hydrate.2. Immerse in orthodianisidine.

5 min

3. Wash with tap water.4. Immerse in veronal buffer.

3

×

2 min

5. Immerse in H acid.

5 min

6. Wash with tap water.7. Dehydrate.8. Mount.

❑

Results

Proteins, especially those associated withnucleic acids, are purple or brown stained.

4.1.2.2.3

C

ONTROL

METHODS

FOR

TETRAZOREACTION

Tetrazoreaction permits visualization of histidine,tryptophan, tyrosine, lysine, cysteine, and arginine.The use of clamping methods for some of theseamino acids permits more precise characterization.

Clamping with performic acid—

Performicacid is used to clamp tryptophan

❑

Reagents

�

Performic acid:—Formic acid 98%

40 mL

—Hydrogen peroxide 30%

4 mL

—Concentrated sulfuric acid

0.5 mL

�

Let rest

1 h

❑

Protocol

1. Dewax, hydrate.2. Immerse in performic acid.

20 min

3. Wash with tap water.4. Tetrazoreact.

Results

Tetrazoreaction is positive for tyrosine, histi-dine, lysine, cysteine, arginine.

Clamping with dinitrofluorobenzene(DNFB)—

This molecule clamps tyrosine, histi-dine, cysteine, lysine.

❑

Reagents

�

DNFB saturated in sodium carbonate satu-rated in ethanol 90%

❑

Protocol

1. Dewax.2. Immerse in ethanol 90%.

10 min

3. Immerse in DNFB.

24 h

➫

This method provides good vizualization ofproteins, but it is not specific.

➫

This solution must be used within 24 h.

➫

See above.



88

4. Wash with ethanol 90%.5. Wash with tap water.6. Tetrazoreact.

❑

Results

Tetrazoreaction is positive for tryptophan andarginine.

Clamping by benzoylation—

Hydroxyl andamine groups are clamped.

❑

Reagents

�

Benzoyl chloride 10% in anhydrous pyridine.

❑

Protocol

1. Dewax.2. Immerse in ethanol 100%.

10 min

3. Immerse in petrol ether.

3 min

4. Let dry in air.5. Immerse in benzoyl chloride.

12 h

at

25˚C

6. Immerse in acetone.

10 min


10 min

8. Hydrate.9. Tetrazoreact.

❑

Results

Tetrazoreaction is positive for histidine andnucleic acids.

4.1.2.2.4

V

ISUALIZATION

OF

NUCLEIC

ACIDS

1. Clamp by benzoylation.2. Tetrazoreact.

4.1.2.2.5

S

TAINING

OF

SECTION

BOTTOM

The tetrazoreaction alone provides sufficientstaining. The bottom is generally weakly stainedby the diazonium salt used. The main substancesthat are researched are specifically stronglystained.

➫

See above.

➫

See above.

➫

Nucleic acid staining is not only linked tonitrogenous bases, but it is also related to thehistidine belonging to the proteinic part.

➫

See above.

➫

See above.

4.1.3 Pentose Visualization

4.1.3.1 Method of Turchini et al.

4.1.3.1.1

P

RINCIPLE

Turchini’s method is based on a condensationbetween pentoses and 9-phenyl 2, 3, 7-tetrahy-drofluorone. However, the specificity of thismethod has not been perfectly established.Fluorone (or its derivative) is condensed with thepentose after an acidic hydrolysis. The product

➫

It is also possible to use a derivative of thismolecule.


4.1 General Methods

89

obtained yields, at a basic pH, a stained insolu-ble molecule. Methyl derivative gives blue-pur-ple staining for ribose (RNA. and red-orange fordeoxyribose (DNA).

4.1.3.1.2

P

ROTOCOL

❑

Fixatives

All the classic fixatives can be used.

Reagents

�

Hydrochloride acid

M

�

Ethanol 80%

�

Sodium carbonate 1%

�

Fluorone:—9-Methyl-2,3,7-trihydroxy fluorone

0.5 g

—Ethanol 95%

100 mL

—Sulfuric acid

1 mL

❑

Protocol

1. Dewax, hydrate.2. Immerse in hydrochloride acid

M

.

10 min

at

60˚C


15 sec

4. Immerse in fluorone.

12 h

5. Immerse in sodium carbonate 1%.

2 min

6. Rinse in distilled water.

2 min

7. Immerse in acetone 50%. 5 min8. Immerse in absolute acetone. 5 min9. Immerse in xylene – acetone (50:50). 5 min10. Immerse in xylene. 10 min11. Mount.

ResultsDNA is violet-blue stained. RNA is orange-red.

4.1.3.1.3 HYDROLYSIS DURATION

The duration of hydrolysis varies according tothe fixative.

� Bouin’s fluid 7 min� Formalin 7 min� Carnoy’s fluid 8 min� Helly’s fluid 25 min� Zenker’s fluid 10 min

➫Fluorone incubation time can vary accordingto the studied tissue. In the original technique,this time varies from 4 to 14 h. It is necessaryto test each material studied to determine theoptimum time.

➫These times are only indications. It is nec-essary to determine the hydrolysis durationeach time a new material is studied.

4.1.3.2 Other methods

Other methods permit visualization of deoxyri-bose but they are rarely used. The more often usedmethod is certainly the Feulgen and Rossenbecknuclear reaction, which is based on specific dena-turation of deoxyribose and visualization of alde-hyde groups by Schiff’s reagent.

➫The Feulgen and Rossenbeck nuclear reac-tion is detailed in Section 4.3.



90

4.1.4 Visualization of Proteins

Among the methods used to visualize proteinicparts, some are descriptive (Hartig–Zacharias’smethod, for instance) and others are actuallyhistochemical, such as original Danielli’s tetra-zoreaction method.

4.1.4.1 Hartig–Zacharias’s method

This is a descriptive method, not a histochemicalone, but it permits one to visualize proteins spe-cifically.❑ FixativeAll the classic fixatives are convenient, but avoidosmium tetroxide.❑ Reagents

� Nuclear fast red� Potassium ferrocyanide 2% in sodium

chloride 1%� Ferric chloride 1% in distilled water

❑ Protocol1. Dewax, hydrate.2. Immerse in potassium ferrocyanide. 10 min3. Wash with tap water.4. Immerse in ferric chloride. 2 min5. Wash with tap water.6. Immerse in nuclear fast red. 1 min7. Wash with tap water.8. Dehydrate.9. Mount.

❑ ResultsProteins are blue stained. Nucleic acids are red(if nuclear fast red has been used).


➫Ferric chloride is sometimes called “ironperchloride.”

➫Washing between ferrocyanide and ferricchloride is optional.

➫Nuclear fast red staining is facultative andcan be avoided to visualize nucleic acids.

➫This method is useful if quick results mustbe obtained.

4.1.4.2 Danielli’s tetrazoreaction ➫See Section 4.1.2.2.2.

4.1.4.3 T chloramine–Schiff method

This method is based on an oxidative deamina-tion with a liberation of aldehyde groups. Theselatter combine with Schiff’s reagent to yield acharacteristic staining pattern.❑ FixativeAll the classic fixatives are convenient. Avoidosmium tetroxide.❑ Reagents

� Schiff’s reagent


4.1 General Methods

91

� PBS pH 7.5—Disodium phosphate (14.198 g/L)

841 mL—Monosodium phosphate (13.805 g/L)

159 mL� T chloramine 1% in buffer� Sodium thiosulfate (hyposulfite) 5 %� Sulfurous water:—Sodium metabisulfite 10% 10 mL—Distilled water 190 mL

❑ Protocol1. Dewax, collodion, hydrate.2. Immerse in T chloramine. 6 h

at 37˚C3. Rinse quickly with distilled water.4. Immerse in thiosulfate. 3 min5. Rinse with distilled water.6. Rinse with sulfurous water.7. Wash with tap water.8. Dehydrate.9. Mount.

❑ ResultsProteins with terminal –NH2 groups are pinkishstained.


➫Collodion only if it is necessary.

➫Thiosulfate eliminates excess of T chloram-ine.

4.1.4.4 Visualization of proteins byCoomassie blue

❑ FixativeAll the classic fixatives are convenient.

❑ Reagents� Triton X-100 1% in water

� Coomassie blue

❑ Protocol1. Dewax, hydrate.2. Immerse in Triton X-100. 15 min3. Immerse in Coomassie blue. 45 min4. Rinse with distilled water or PBS (pH 7.0).5. Mount in aqueous medium.

➫Coomassie blue is also called “light blue.”

➫Triton X-100 is a detergent that makes phos-pholipid layers permeable. It allows the dye topenetrate. Its use is optional in certain cases.➫See Chapter 7: Preparation of Products.

➫The action of Coomassie blue can beincreased to 60 min.➫It is possible to mount sections with glycerol50% in distilled water.➫This method is classically used to visualizeall the proteins belonging to a tissue.



92

4.2 BASOPHILIC REACTIONS


Nucleic acids are linked to ionized acid groupsthat are present on phosphate entities. At a pHlower than 2, electronegative charges appear.These charges can fix basic dyes. Negative phos-phoric groups are also bound with proteins inlive cells. This fact explains why nucleic acidscannot fix basic dyes in live cells. Preservationfrees these groups, but there is always somecompetition between cations of dye and othercations belonging to the tissue, particularlyamine groups belonging to the proteinic part ofnucleoproteins.

➫Basic dyes, as previously mentioned, can fixthemselves onto other acidic groups that do notbelong to nucleic acids. One often must usedyes with a certain specificity, for example,methyl green or pyronine for DNA and RNA,respectively. But use of dyes with a large spec-trum of possibilities, such as toluidine blue, isnot excluded.➫DNA visualization is relatively easy. ForRNA, control reactions must be performed.

4.2.2 Gallocyanin Method

4.2.2.1 Principle

At high temperature, gallocyanin forms threelack types with chrome alum. One of them iscationic and fixes itself onto phosphate groupsbelonging to nucleic acids. This forms a darkblue complex, which is obtained at a pH varyingfrom 0.8 to 4.2. However, to prevent this sub-stance from fixing onto other molecules, oper-ating at a pH ranging from 1.50 to 1.75 isrecommended. In these conditions, the stain ishighly selective for nucleic acids.


Figure 4.2 Gallocyanin.

4.2.2.3 Method

❑ Fixatives ➫If possible, avoid fixation with osmium

COOH

N

(CH3)2N O O


4.2 Basophilic Reactions

93

All classic fixatives can be used.

❑ Reagent� Gallocyanin chromic lac

❑ Protocol1. Dewax, hydrate.2. Immerse in gallocyanin lac. 24 h3. Running tap water. 5 min4. Dehydrate.5. Mount.

❑ ResultsDNA and structures with basophilic compo-nents are dark blue stained.

t e t r ox ide and fix ing s ec t i ons a f t e rpostchromization.


➫The gallocyanin immersion time can beincreased.

➫Other dyes can be added to gallocyanin, forexample, picrofuchsin.

4.2.3 Methyl Green Method

4.2.3.1 Principle

This method allows visualization of DNA alone.Methyl green stains DNA if the DNA is notdepolymerized. Other basophilic substances,such as mucopolysaccharides or cartilaginoussubstances, are also stained.


Figure 4.3 Methyl green.

4.2.3.3 Method

❑ FixativeCarnoy’s fluid is recommended, but numerousother fixative fluids can be used.

❑ Reagents� Methyl green

❑ Protocol1. Dewax, hydrate.2. Immerse in methyl green. 10 min3. Dry the slide on a paper filter.

➫Practically all the classic fixatives are con-venient, but a short preservation to avoidnucleic acid depolymerization is recom-mended. Only a few hours are necessary.➫See Chapter 7: Preparation of Products.

2 CI-

N(CH3)2

C

N+(CH3)2

(CH3)3N+



94

4. Dehydrate slides with butanol in twoquick baths.5. Immerse in cyclohexane. 10 min6. Mount.

❑ ResultsDNA is green stained. Mucopolysaccharides canalso be green stained.

➫Chromatin details are very well visualized.However, only nuclear staining permits thepresence of DNA to be certified.

4.2.4 Pyronine Method

4.2.4.1 Principle

Pyronine is a basic dye that is highly RNA spe-cific. It is generally used in the Pappenheim–Unna staining method, associated with methylgreen. It can also be used alone to visualizeRNAs.

➫The deletion of the stain after ribonucleaseaction shows that it has actual specificity.


Figure 4.4 Pyronine.

4.2.4.3 Method

❑ FixativeOf the fixatives, Bouin’s fluid should be avoidedbecause it depolymerizes DNA.

❑ Reagent� Pyronine

❑ Protocol1. Dewax, hydrate.2. Immerse in pyronine. 10 min3. Dry slide with filter paper.4. Dehydrate slides with two quick dips inbutanol.5. Immerse in cyclohexane. 10 min6. Mount.

❑ ResultsRNA is red stained.

➫See Section 4.2.3.3. Practically all the classicfixatives are convenient, but a short preserva-tion to avoid nucleic acid depolymerization isrecommended. Only a few hours are necessary.➫See Chapter 7: Preparation of Products.

➫This stoichiometric method is very useful toappreciate all RNA molecules in cells. It canbe useful in studying the evolution of RNAduring embryonic development or as a patho-logical phenomenon.

(CH3)2N O N +(CH 3) 2

CI-



95

4.2.5 Pappenheim–Unna Staining

4.2.5.1 Principle

This staining technique is also called methylgreen pyronine staining. It permits one to stainsimultaneously, and in a differentiated man-ner, RNA molecules in red and DNA in green.Basic dyes, which are positively charged, mixwith the negative components (acids) that arebeing investigated, in this case, nucleic acids.If the tissues are submitted to two basic dyes,the coloration obtained will depend on theaffinity of the tissue or cell molecule for thesedyes. These affinities depend on pH.In the present case, for a pH between 4 and 5,methyl green will stain DNA, and pyronine willstain RNA molecules. The addition of both dyesinto nuclei will stain them purple.At pH 1.5, methyl green alone reacts and DNAalone is stained. At pH 9.3 and greater, pyroninealone reacts and RNA alone is stained.

➫Caution! Although pyronine is an RNA-spe-cific dye, it differs from methyl green,whichalso reacts with mucous substances, masto-cytes, granulations, and sulfate chondroitins incartilage. However, nuclear staining willalways permit one to verify the presence ofDNA.

4.2.5.2 Method

❑ FixativeCarnoy’s fluid is recommended. Acidic fixativesmust be avoided. However, such fixatives as for-malin or Bouin’s fluid can be used.

❑ Reagents� Methyl green pyronine

❑ Protocol1. Dewax, hydrate.2. Immerse in methyl green pyronine.

10 min3. Dry slide on filter paper.4. Dehydrate slide by two quick dips inbutanol.5. Immerse in cyclohexane. 10 min6. Mount.

❑ ResultsNucleic DNA and RNA are purple-blue stained.In cytoplasm, RNA is pinkish. Acidic muco-polysaccharides can be purple stained.

➫See Section 4.2.3.3. Practically all the classicfixatives are convenient, but short preservationis recommended to avoid nucleic acid depoly-merization. Only a few hours are necessary.


➫The Pappenheim–Unna staining method isvery useful to appreciate the evolution ofnucleic acids during the differentiation ofembryonic tissues or the evolution of certaincell types (sperm, for instance).



96

4.2.6 Controls

4.2.6.1 Ribonuclease Brachet’s test


❑ Reagents� Crystallized ribonuclease 0.01% in dis-

tilled water� Methyl green pyronine

❑ Protocol1. Prepare three groups of deparaffined slideswhich are neither collodioned nor hydrated.2. Treat one group with ribonuclease. 1 h

at 37˚C3. Wash with tap water.4. Treat the second group with distilledwater. 1 h

at 37˚C5. Stain the three groups with methyl greenpyronine.

❑ ResultsA pure green staining of DNA must be observedon the slide treated with ribonuclease.

➫See Section 4.2.3.3. Practically all the classicfixatives are convenient, but a short preserva-tion is recommended to avoid nucleic aciddepolymerization. Only a few hours are nec-essary.


➫See Section 4.2.3.

➫Brachet’s test with ribonuclease is an essen-tial step in nucleic acid visualization with Pap-penheim–Unna staining.

4.2.6.2 RNA extraction by hydrochloricacid


❑ Reagents� Normal solution of hydrochloric acid

❑ Protocol1. Prepare three groups of deparaffinedslides which are neither collodioned norhydrated.2. Treat one group with hydrochloric acid.

10 minat 60˚C

➫See Section 4.2.2.3. Practically all the clas-sic fixatives are convenient, but a short pres-ervation is recommended to avoid nucleicacid depolymerization. Only a few hours arenecessary.



97

3. Treat the second group with distilledwater. 10 min

at 60˚C

4. Stain the three groups with methyl greenpyronine.

❑ ResultsA pure green staining of DNA will be observedon the slide treated with ribonuclease.

➫See Section 4.2.3.

4.2.7 Mann–Dominici’s Staining

The Mann–Dominici staining method is not gen-erally used as a histochemical method; however,it is based on the use of a basic and metachro-matic dye, toluidine blue. This method can beuseful to detect nucleic acids.

❑ FixativeAll classic fixatives are convenient, but oxidiz-ing fixatives containing potassium dichromateor osmium tetroxide should be avoided.❑ Reagents

� Erythrosine-G orange� Toluidine blue� Potassium permanganate (KMnO4) in aque-

ous solution 0.25%� Sodium bisulfite (or metabisulfite) 2%� Acetic water 0.25%

❑ Protocol1. Dewax, hydrate.2. Immerse in potassium permanganate. 30 s3. Rinse with distilled water.4. Immerse in sodium bisulfite. 1 min5. Wash with tap water.6. Immerse in erythrosine-G orange.

10 min7. Rinse with distilled water.8. Immerse in toluidine blue. 1 min9. Rinse with distilled water.

10. Immerse in acetic water.



➫This method is useful to gauge specific his-tochemical reactions to analyze tissue compo-nents.➫This staining method is difficult to performsuccessfully because there are two differentia-tion stages and care is required.

➫See Chapter 7: Preparation of Products.➫See Chapter 7: Preparation of Products.➫Potassium permanganate solution is pre-pared from a stock solution at 2.5%.

➫Immerse slides in acetic water to obtain gen-eralized purple staining. Control under amicroscope.➫At this stage, toluidine blue differentiationcontinues. Stop the reaction under microscopecontrol.➫Ethanol 100% stops the differentiation.



98

13. Continue to dehydrate.14. Mount.

❑ ResultsNuclei, basophilic cytoplasm, and severalsecretions are purple-blue stained. Acidophiliccytoplasm, nucleoli, and several secretions arepinkish. Proteinic secretions and pigments aregreen-blue. Metachromatic mucus is purplestained.

4.2.8 Semi-thin Section Staining with Toluidine Blue

See Section 1.6 for visualization of nucleic acidsusing general methods.

4.2.9 Love and Liles’s and Love and Suskind’s Methods

4.2.9.1 Principle

Phosphoric groups that are bound to nucleopro-tein amine groups are released by nitrous acidor formaldehyde. Toluidine blue is then fixed onthese phosphoric groups. Toluidine blue thenreacts with molybdate, yields a metachromaticreaction with color varying as a function of thenature of the nucleic acid (DNA or RNA).

4.2.9.2 Methods for paraffin sections

❑ FixativeSublimated formalin is recommended.

❑ Reagents� Lugol� Sodium hyposulfite 5 %� Nitrous acid� Toluidine blue 0.01%� Ammonium molybdate 15% and 5%� Sublimated formalin

❑ Protocol1. First group of slides

a. Dewax, hydrate.

➫The reaction is based on comparing theresults obtained with sections submitted tosublimated formalin and those not submittedto it (mordant) and the results of those submit-ted to nitrous deamination.

➫The first group of sections gives results fortissues that are deaminated by nitrous acid.



99

b. Rinse with tap water. 5 minc. Immerse in lugol. 5 mind. Immerse in sodium hyposulfite. 5 mine. Rinse with tap water. 5 minf. Immerse in nitrous acid. 18 hg. Rinse with tap water. 10 sh. Immerse in toluidine blue 0.01%.30 mini. Immerse in ammonium molybdate 15%.

30 minj. Rinse with tap water. 10 sk. Dehydrate.

l. Mount.

2. Second group of sections

a. Dewax, hydrate.b. Immerse in sublimated formalin 2, 3 and 4 hc. Rinse with tap water. 5 mind. Immerse in lugol. 5 mine. Immerse in sodium hyposulfite. 5 minf. Rinse with tap water. 5 ming. Immerse in toluidine blue 0.01%.30 minh. Rinse with tap water. 10 si. Ammonium molybdate 15%. 30 minj. Rinse with tap water. 10 sk. Dehydrate.

m. Mount.

3.Third group of sectionsa. Dewax, hydrate.b. Rinse with tap water. 5 minc. Immerse in lugol. 5 mind. Immerse in sodium hyposulfite. 5 mine. Rinse with tap water. 5 minf. Immerse in toluidine blue 0.01%. 30 ming. Immerse in ammonium molybdate 5%.

15 minh. Rinse with tap water. 10 si. Dehydrate.

j. Mount.

❑ ResultsDNA and RNA molecules are stained at varyingintensities by toluidine blue. Controls can be done

➫Dehydration must be done with tertiarybutanol (2-methylpropane-2-ol).➫Mounting with Permount medium is recom-mended.➫The second group of sections gives resultsfor tissues that are deaminated by formalde-hyde.

➫Dehydration must be done with tertiarybutanol (2-methylpropane-2-ol).➫Mounting with Permount medium is recom-mended.➫The third group of sections gives results fortissues that are not deaminated.

➫Dehydration must be done with tertiarybutanol (2-methylpropane-2-ol).➫Mounting with Permount medium is recom-mended.

➫In the original method, the authors distin-guish three staining stages: Stage I staining is



100

by ribonuclease action or by comparison withother staining methods, such as the Feulgenreaction.

given without deamination. Stage II staining isgiven after sublimated formalin deaminationfor 2, 3, or 4 h. Stage III is given after nitrousacid action.

4.2.9.3 Method for smears

❑ FixativeSublimated formalin is recommended.❑ Reagents

� Lugol� Sodium hyposulfite� Nitrous acid� Toluidine blue 0.004% and 0.01%� Ammonium molybdate 15%� Sublimated formalin

❑ Protocol1. First smear

a. Immerse in sublimated formalin 1 hat 37˚C

b. Rinse with tap water 5 minc. Immerse in lugol 5 mind. Immerse in sodium hyposulfite 5 mine. Rinse with tap water 5 minf. Immerse in nitrous acid 18 hg. Rinse with tap water 10 sh. Immerse in toluidine blue 0.004%

30 mini. Immerse in ammonium molybdate 15%

30 minj. Rinse with tap water 10 sk. Dehydrate

l. Mount

2. Second smeara. Immerse in sublimated formalin.

5 and 10 minat 37˚C

b. Rinse with tap water. 5 minc. Immerse in lugol. 5 mind. Immerse in sodium hyposulfite. 5 mine. Rinse with tap water. 5 minf. Immerse in toluidine blue 0.01%. 30 ming. Rinse with tap water. 10 sh. Immerse in ammonium molybdate 15%.

30 mini. Rinse with tap water. 10 s

➫The first smear gives results for tissues thatare deaminated by nitrous acid.


➫The second smear gives results for tissuesthat are deaminated by formaldehyde.


4.3 Feulgen and Rossenbeck Nuclear Reaction

101

j. Dehydrate.

k. Mount.

3. Third smeara. Immerse in sublimated formalin. 1 and 2 h

at 37˚Cb. Rinse with tap water. 5 minc. Immerse in lugol. 5 mind. Immerse in sodium hyposulfite. 5 mine. Rinse with tap water. 5 minf. Immerse in toluidine blue 0.01%. 30 ming. Rinse with tap water. 10 sh. Immerse in ammonium molybdate 15%.

30 mini. Rinse with tap water. 10 sj. Dehydrate.

k. Mount.

❑ ResultsDNA and RNA molecules are stained with varyingintensity by toluidine blue. Controls can be doneby ribonuclease action or by comparison with otherstaining methods, such as the Feulgen reaction.

➫Dehydration must be done with tertiarybutanol (2-methylpropane-2-ol).➫Mounting with Permount medium is recom-mended.➫The third smear gives results for tissues thatare not deaminated.


4.3 FEULGEN AND ROSSENBECK NUCLEAR REACTION

4.3.1 Principle


“Nuclear reaction” should be distinguished from“plasmal reaction.” The Feulgen and Rossenbecknuclear reaction was published in its definitiveform in 1924. The method consists of submittingDNA to an acidic hydrolysis that exclusivelyreacts with puric bases and deoxyribose binding.This releases aldehydes, which are then stainedby Feulgen’s reagent or a similar substance. Thereaction is done in two steps: first, hydrolysis ofDNA to release an aldehyde and sugars and, sec-ond, detection of the aldehyde by a “Feulgen–Schiff” reagent type. This method is DNA spe-cific and cannot be applied to RNA.

➫The Feulgen and Rossenbeck plasmal reac-tion is a method used to visualize acetalphos-phatide molecules (lipids) in the cytoplasm ofsome cell types.



102

4.3.1.2 Chemical reaction of acidic hydrolysis

The chemical reactions are not precisely knownbut one mechanism is currently accepted (Fig-ure 4.5).

➫The hydrolysis only affects deoxyribosebound to a purine base, which yields a DNA-specific hydrolysis.

Figure 4.5 Feulgen hydrolysis reaction.

4.3.1.3 Schiff staining ➫Unstained or pale-yellow stained Schiff’sreagent reacts with aldehyde to yield a red-stained product (Figure 4.6).

Figure 4.6 Feulgen’s reaction.

4.3.2 Schiff’s Reagent

Schiff’s reagent, which is also called Schiff’sleucofuchsin, is a mixture of basic fuchsin andsulfuric acid (Figure 4.7).

P

P

P

P

P

P

CH2

CH2

CH2

CH2

COH

COH

CH2

CH2

O

O

O

O

Purine

Pyrimidine

Purine

Purine

Pyrimidine

Purine

HYDROLYSIS

SO 3H

C

H 2SO 1

N CHR

CH

R

R

N CHR

SCHIFF + 2 RCHO H 2N

CHNH

C

SO 3

NH + SO 3-

H 3N -

CH

R

R

CHNH

C +

SO 3-

NH SO 3

H 3N +



103

❑ Preparation

❑ ResultA pale-yellow fluid must be obtained.

Figure 4.7 Schiff’s reagent.


➫Several forms of perfectly uncolored Schiff’sreagent are available.

4.3.3 Histochemical Practice

4.3.3.1 Preservation

Tissue preservation for visualizing DNA bythe Feulgen and Rossenbeck method is partic-ularly important because the reaction protocoldepends on the nature of the fixative. Thefixative must provide the best morphological,cytological, or histological preservation pos-sible. But, on the other hand, the duration ofthe hydrolysis, which is fundamental to visu-alization of DNA, also depends on the natureof the fixative. Certain fixatives, such as acidicfluids, must be avoided. They induce thebeginning of hydrolysis. If hydrolysis is toostrong, DNA molecule degradation will con-tinue beyond separation of puric bases anddeoxyribose. That will induce total degrada-tion of the DNA molecule, making it impos-sible to visualize.Numerous studies concern the fixative and pHeffects. It is now possible to realize a Feulgenand Rossenbeck reaction on tissues preservedwith very different fixatives. Therefore, it is nec-essary to modulate the duration of the hydro-chloric acid action.

➫Studies dealing with the effects of preserva-tion have been done with Bouin’s fluid. Thisfixative must be avoided because it is acidicand can inhibit the Feulgen and Rossenbeckreaction. However, the true effects of this fix-ative are not known. According to Gabe(1968), Bouin’s fluid could induce a high levelof DNA polymerization by binding withhydrolysis sites. This effect would preventhydrochloric acid action from liberating alde-hyde groups. On the contrary, according to Ganter and Jolles(1969), Bouin’s fluid begins to hydrolyze DNAmolecules that would then be stronglydegraded by a too lengthy acidic hydrolysis.However, it is still possible to have good resultswith Bouin’s fluid preservation.

NH2 NH2 NH2 NH2

SO3HH2SO2

NH2+CL- NH3

-CL-

+C C



104

4.3.3.2 Hydrolysis

❑ DurationIn the classic method, acidic hydrolysis is donewith hydrochloride acid M at 60˚C. The durationof hydrolysis is an essential factor in obtaininggood results. If the hydrolysis duration is veryshort, puric base and deoxyribose separation isincomplete and aldehydes are not released orthey are released in very tiny quantities. If thehydrolysis duration is very long, DNA depoly-merization continues. Molecules with aldehydefunctions are lost because, separated from thewhole molecule, they go into solution and areweakly stained. In some cases, there is no reac-tion. An optimal duration must be strictlyobserved for the hydrolysis.

❑ Duration of hydrolysis as a function of the fixative� Bouin’s fluid 2 min� Champy’s fluid 25 min� Carnoy’s fluid 8 min� Formalin 8 min� Sublimated formalin 8 min� Ethanol 100% 5 min� Flemming’s fluid 16 min� Helly’s fluid 8 min� Heidenhain Susa 18 min� Zenker’s fluid 5 min

❑ TemperatureAcidic reaction is generally done at 60˚C toaccelerate the reaction. However, several authorshave tried to modify this protocol to limit theeffects of high temperature on the sections.❑ Alternative methodsHydrolysis can be done with hydrochloric acidin ethanol 100% solution, at 60˚C. In this case,the small DNA molecules and their binding withproteins are preserved.In another method, hydrolysis is done at roomtemperature, with hydrochloric acid 5 M. It isalso possible to proceed to a slow hydrolysisusing hydrochloric acid at pH 1.2 and at 37˚C.❑ Other acidsAcids other than hydrochloric acid can be used:citric acid, perchloric acid, phosphoric acid,chromic acid (chrome trioxide), or sulfuric acid.

➫It is also possible to use hydrochloric acid 5M at room temperature (20˚C). This methodcan be very useful to obtain very precise visu-alization of chromatin details.

➫When an organ is studied for the first time,it is necessary to conduct a series of tests todetermine the optimal duration of hydrolysis.When a Feulgen and Rossenbeck reaction isrigorously done, results can be studied byquantitative analysis, because the reaction isstoichiometric.

➫It is also possible to use low-temperaturehydrolysis.



105

❑ Stopping the reactionThe reaction can be stopped with cold water.

4.3.4 Protocol

4.3.4.1 General protocol

❑ FixativeCarnoy’s or Flemming’s fluids are often recom-mended but it is possible to use numerous otherfixatives. It is necessary to determine the optimalduration of hydrolysis for each.❑ Reagents

� Schiff’s reagent� Hydrochloric acid M� Sulfurous water—Sodium metabisulfite 10% 10 mL—Distilled water 190 mL

❑ Protocol1. Dewax, hydrate.2. Immerse in hydrochloric acid. 8 min

at 60 ˚C3. Rinse with tap water. 8 min4. Immerse in Schiff’s reagent. 1 h5. Rinse with tap water. 5 min6. Rinse in sulfurous water. 3 × 1 min7. Rinse with tap water. 5 min8. Dehydrate.9. Mount.

❑ ResultsDNA is red stained.

➫For determination of hydrolysis duration,see Section 4.3.3.2.


➫The time given is for tissues preserved withCarnoy’s fluid. The duration may differ withanother fixative.

➫The Feulgen and Rossenbeck reaction is sto-ichiometric and thus useful for DNA quantifi-cation by automatic methods.

4.3.4.2 First variant

❑ FixativeCarnoy’s or Flemming’s fluids are often recom-mended, but it is possible to use numerous otherfixatives. It is necessary to determine the optimalduration of hydrolysis for each.❑ Reagents

� Schiff’s reagent� Hydrochloric acid M� Sulfurous water:—Sodium metabisulfite 10% 10 mL—Distilled water 190 mL

➫For determination of hydrolysis duration,see Section 4.3.3.2.



106


at 60 ˚C3. Rinse with tap water. 8 min4. Immerse in Schiff’s reagent. 1 h5. Rinse with tap water. 5 min6. Rinse in sulfurous water. 3 × 1 min7. Rinse with tap water. 5 min8. Immerse in picro indigo carmine. 30 s9. Rinse with tap water. 5 min10. Dehydrate directly with ethanol 100%.11. Mount.

❑ ResultsDNA is red stained. Acidophilic cytoplasm isyellow or green; collagen is blue; red blood cellsare yellow stained; and glycoproteins are brown.Secretions can be yellow or green stained.


➫The Feulgen and Rossenbeck reaction is sto-ichiometric; bottom staining (cytoplasm, col-lagen, and cell inclusions) can hinderautomatic quantitative analysis of DNA.

4.3.4.3 Second variant

❑ FixativeCarnoy’s or Flemming’s fluids are often recom-mended, but it is possible to use numerous otherfixatives. It is necessary to determine the optimalduration of hydrolysis for each.❑ Reagents

� Schiff’s reagent� Hydrochloric acid 5 M� Sulfurous water—Sodium metabisulfite 10% 10 mL—Distilled water 190 mL

❑ Protocol1. Dewax, hydrate.2. Immerse in hydrochloric acid. 8 min3. Rinse with tap water. 5 min4. Immerse in Schiff’s reagent. 1 h5. Rinse with tap water. 5 min6. Rinse in sulfurous water. 3 × 1 min7. Rinse with tap water. 5 min8. Immerse in picro indigo carmine. 30 s9. Rinse with tap water. 5 min10. Dehydrate directly with ethanol 100%.11. Mount.

❑ ResultsDNA is red stained. Acidophilic cytoplasm isyellow or green; collagen is blue; red blood cells

➫For determination of hydrolysis duration,see Section 4.3.3.2.➫See Chapter 7: Preparation of Products


➫The Feulgen and Rossenbeck reaction isstoichiometric and thus useful for DNA



107

are yellow stained; and glycoproteins are brown.Secretions can be yellow or green stained.

quantification by automatic methods. However,bottom staining can hinder quantification.

4.3.5 Alternative Methods to Feulgen’s Reaction

4.3.5.1 General principles

Some methods based on the same principlecan replace the original Feulgen and Rossen-beck reaction. All these methods are based onan acidic hydrolysis that separates puric basesand deoxyribose, releasing aldehyde groups.The difference between the reactions is gen-erally linked to the methods used to visualizealdehydes.

4.3.5.2 Thionin–SO2 method

In this technique, a 0.25% thionin–SO2 solutionis used to replace Schiff’s reagent.❑ FixativeCarnoy’s or Flemming’s fluids are recom-mended.

❑ Reagents� Thionin

� Hydrochloric acid M� Sulfurous water—Sodium metabisulfite 10% 10 mL—Water 190 mL� Van Gieson’s picrofuchsin

❑ Protocol1. Dewax, hydrate.2. Immerse in hydrochloric acid. 4 min3. Rinse with tap water. 4 min4. Immerse in thionin–thionin chloride. 1 h5. Rinse with sulfurous water.6. Immerse in Van Gieson’s picrofuchsin.

30s7. Dehydrate directly with ethanol 100%.8. Mount.

❑ ResultsDNA is deep blue stained. Acidic substances aremetachomatic.

➫This method is also called De Lamater’smethod.➫Thionin–SO2 can be replaced with A azure-SO2.

➫See Chapter 7: Preparation of Products.➫Before use, add one drop of thionin chloridefor 10 mL thionin or 5 mL A azure.


➫This method is stoichiometric. The blue-stained DNA can be easily quantified by auto-matic image analysis.



108

4.3.5.3 Himes and Moriber’s method

In this method, nuclear reaction and PAS areassociated.❑ FixativeCarnoy’s or Flemming’s fluids are recommended.❑ Reagents

� A azure–SO2 � Whiting fluid—Sodium metabisulfite 5% 5mL—Hydrochloric acid M 5 mL—Distilled water 90 mL� Hydrochloric acid M� Periodic acid� Schiff’s reagent� Naphthol yellow S


at 60˚C3. Wash with distilled water.4. Immerse in A azure–SO2. 5 min5. Rinse with distilled water.6. Immerse in whiting solution. 2 × 2 min7. Immerse in periodic acid. 2 min8. Rinse with distilled water.9. Immerse in Schiff’s reagent. 2 min10. Immerse in whiting solution. 2 × 2 min11. Immerse in naphthol yellow S. 2 min12. Rinse with tap water.13. Dry with filter paper.14. Dehydrate.15. Mount.

❑ Results Nuclei are blue or green stained. PAS positivecomponents are red and basic proteins are yellow.

➫See Chapter 7: Preparation of Products.➫Prepare at time of use.


➫Use of tertiary butanol (2-methylpropane-2-ol) is recommended.

4.3.5.4 Benson’s method

❑ FixativeCarnoy’s or Flemming’s fluids are recommended.❑ Reagents

� A azure–SO2� Alcian blue pH 2� Hydrochloric acid 5 M and 0.01 M� Periodic acid� Schiff’s reagent� Naphthol yellow S� Acetic acid 1%

❑ Protocol1. Dewax, hydrate.

➫In the original method, preservation is donewith formalin 10% added to 0.5% cetylpyri-dinium chloride.➫See Chapter 7: Preparation of Products.➫See Chapter 7: Preparation of Products.




109

2. Immerse in hydrochloric acid. 9 minat RT

3. Rinse with tap water. 1 min4. Immerse in A azure–SO2. 10 min5. Immerse in whiting solution. 2 × 2 min6. Rinse with tap water. 1 min7. Immerse in alcian blue. 10 min8. Hydrochloric acid 0.01 M. 1 min

9. Rinse with tap water. 1 min10. Immerse in periodic acid. 5 min11. Rinse with tap water. 1 min12. Immerse in whiting solution. 2 x 2 min13. Rinse with tap water. 1 min14. Immerse in naphthol yellow S. 2 min15. Immerse in acetic acid 1%. 2 min16. Dry with paper filter.17. Dehydrate .18. Mount.

❑ Results Nuclei are blue or green stained. PAS-positivecomponents are red, basic proteins are yellow,and acidic carbohydrates are blue.

➫Washing can be prolonged.


➫Hydrochloric acid hydrolysis must be pre-cisely adapted to avoid interferences betweenDNA blue staining by azure A and the bluealcian staining.➫Washing can be prolonged.



4.3.5.5 Silver methenamine method

4.3.5.5.1 PRINCIPLE

With a reducing substance, silver methenamine isreduced with metal silver deposit on the reducinggroup. For DNA visualization, the aldehyde groupobtained after acidic hydrolysis is the reducingsubstance, and it is visualized by a deposit of silver.During the hydrochloric acid hydrolysis that isused for the Feulgen and Rossenbeck reaction,deoxyribose is diffused even before the reducingreaction. It would be difficult to obtain a con-clusive result. To avoid this diffusion, the reac-tion is done with citric acid.Aldehydes are visualized by silver and are blackstained.

4.3.5.5.2 Protocol❑ FixativeNeutral formalin and Carnoy’s fluid are used onfrozen sections, smears, and chromosome prep-arations.

➫This method is also called Korson’s method.

➫Silver methenamine can be replaced withother silver salts, such as tetramine hexaethyl-ene.

➫These silver salts can also be used to visu-alize glucids by the PAS method during whichaldehyde groups are formed from glycolsbelonging to the glucid molecule.

➫The precision of the black staining is verystrong. This method can be used to visualizeDNA in transmission electron microscopy.

➫All the usual fixatives are convenient.



110

❑ Reagents� Citric acid M � Silver methenamine� Gold chloride

❑ Protocol1. Dewax, hydrate.

2. Immerse in citric acid 1 M. 30 minat 60 ˚C

3. Rinse with distilled water. 5 min4. Immerse in silver methenamine. 1 h

at 60 ˚C5. Rinse with distilled water.6. Immerse in gold chloride 0.2%. 5 min7. Rinse with distilled water.8. Dehydrate.9. Mount.

❑ ResultsDNA is black stained.

➫Warm the citric acid before each use.➫See Chapter 7: Preparation of Products.

➫For frozen sections and smears, beginhydrolysis directly with citric acid.➫The reaction duration can vary as a functionof the fixative. It is necessary to conduct aseries of tests to determine the optimal dura-tion.

➫The use of gold chloride is optional.

4.4 OTHER REACTIONS


Several methods have been established to visualizenucleic acids, especially DNA. One of them, theperiodic acid and silver diamine method (TheAdams, Bayliss, and Weller method), allows DNAto be visualized specifically.Another method, very different from the first,consists of using the affinity of actinomycin forDNA.

4.4.2 Periodic Acid and Silver Diamine Method

4.4.2.1 Principle

This method is based on DNA visualization bymeans of a silver salt. The mechanism is notknown, but it seems to be DNA specific. It isbased on reduction of a silver diamine by for-malin after periodic oxidization of DNA.


4.4 Other Reactions

111

4.4.2.2 Protocol

❑ FixativeNeutral formalin 10%, Baker formalin calcium.Frozen sections.❑ Reagents

� Ammonium silver nitrate—Ammonium 28% 10 mL—Silver nitrate 80 mL—Ammonium silver nitrate is continuously

poured to dissolve the brown precipitate—Distilled water to dissolve opalescent

solution� Formalin 1%� Gold chloride 0.2%

❑ Protocol1. Immerse in periodic acid. 10 min2. Wash with tap water. 5 min3. Immerse in ammonium silver nitrate.

3 min4. Rinse with distilled water.5. Immerse in formalin 10%. 10 min6. Immerse in gold chloride 0.2%. 2 min7. Rinse with tap water. 5 min8. Mount in aqueous medium.

❑ ResultsDNA is black stained.

➫Adding silver nitrate producess a brown pre-cipitate.

➫Washing must be done as quickly as possible.➫Formalin is used to reduce sites belongingto DNA.

4.4.3 Radioactive Actinomycin

DNA has an affinity for actinomycin. Themethod consists of reacting sections with acti-nomycin containing tritium (3H) or radioactivecarbon (14C). Then, a histoautoradiography isperformed.

➫The specificity of this method has been dem-onstrated by deoxyribonuclease action. In thiscase, no staining is observed.➫If histones are eliminated by trypsin, stain-ing is intensified.

4.4.4 Quantification

DNA quantification is used after a Feulgen andRossenbeck reaction, with Schiff’s reagent, orafter thionin or azure A–SO2 methods. It is alsopossible to quantify DNA after silver diaminestaining or radioactive actinomycin by countingsilver grains.

➫RNA quantification is possible by using astoichiometric reagent, such as pyronine.


Chapter 5

FluorescentMethods


Contents

115

Contents

5.1 Fluorescent Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.1.1 Use of Fluorescent Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.1.2 Fluorescent Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.1.2.1 Intercalating Fluorescent Dyes . . . . . . . . . . . . . . . . . . . . . . . . .5.1.2.2 Feulgen–Schiff-Like Fluorescent Dyes . . . . . . . . . . . . . . . . . . .5.1.2.3 Base Pair-Specific Fluorescent Dyes . . . . . . . . . . . . . . . . . . . . .5.1.2.4 Advantages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2 Staining Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.1 Orange Acridine Staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2.1.1 Mechanism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.1.2 Formula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.1.3 Effects of External Factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.1.4 Action. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.1.5 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2.2 Coriphosphine O Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.2.1 Mechanism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.2.2 Formula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.2.3 Action. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.2.4 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2.3 Propidium Iodide Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.3.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.3.2 Formula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.3.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2.4 Hoechst 33258 Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.4.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.4.2 Formula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.4.3 DNA Visualization by Hoechst 33258 . . . . . . . . . . . . . . . . . . . .5.2.4.4 Visualization of Mycoplasma by Hoechst 33258 . . . . . . . . . . .

5.2.5 Hoechst 33342 Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.5.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.5.2 Formula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.5.3 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2.6 Quinacrine Mustard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.6.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5.2.6.2 Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2.7 DAPI and DIPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

117117117117117117118118118118118118118119119119120120120121121121121121121122122122123123124124124124124125


5.1 Fluorescent Methods

117

5.1 FLUORESCENT METHODS

5.1.1 Use of Fluorescent Dyes

Nucleic acids can be visualized by use of fluo-rochromes. Some of these methods have beenperfected for examining nucleic acids with pho-tonic microscopes. This chapter discusses onlya few of these staining methods.

➫

The development of flux cytometry neces-sitated the use of numerous fluorescent dyes.Their use has often been adapted for micro-scopic examination.

5.1.2 Fluorescent Dyes

Several types of fluorescent dyes can be definedas a function of their association with a nucleicacid.

5.1.2.1 Intercalating fluorescent dyes

These molecules are fixed between the two DNAstrands or within an RNA loop. Orange acridine,ethidium bromide, ethidium chloride, or bro-mide, and coriphosphine O belong in this cate-gory.

5.1.2.2 Feulgen–Schiff-like fluorescent dyes

Several fluorescent dyes can be used, such asSchiff’s reagent, on aldehyde functions that areobtained after DNA acidic hydrolysis. Ariflevinand auromicin belong in this category.

5.1.2.3 Base pair-specific fluorescent dyes

Several fluorescent dyes react with specific basepairs. Some of them are specifically intercalatedbetween guanine and cytosine, for example,DAPI, DIPI, and Hoechst 33258. These dyes areDNA specific.Others intercalate between adenine and thymine.They are fluorescent antibiotic molecules, suchas chromomycin A3, mithramycin, or evenolivemycin.

➫

DAPI: 4

'

,6-diaminido-2-phenylindole

➫

DIPI: 4

'

,6-diaminido-2-imidazolinyl-

4

H-

5

H


Fluorescent Methods

118

5.1.2.4 Advantages

One of the advantages of using a fluorescent dyeis to achieve stoichiometric staining. In certaincases, these dyes allow one to visualize nucleicacids that cannot be observed with other methods.

➫

The use of Hoescht dye permits visualiza-tion of nucleic acids belonging to mycoplasma,when they infect cell cultures.

5.2 STAINING METHODS

5.2.1 Orange Acridine Staining

5.2.1.1 Mechanism

Orange acridine is a basic dye belonging to theacridine family. It lends a green fluorescence tolive cells and a red fluorescence to dead cells.Several studies have shown a variability of flu-orescence that is associated with the fixative.In preserved cells, the nucleus is orange redstained, and the cytoplasm is slightly red. Theother structures are green or yellow fluorescent. On fresh tissue, DNA is green fluorescent andnucleus and cytoplasmic RNA is red. Otherstructures can also be red stained (Figure 5.1).

➫

On fresh tissue, RNase action prevents redfluorescence.

5.2.1.2 Formula

Figure 5.1 Orange acridine.

5.2.1.3 Effects of external factors

The method depends upon pH and dye solutionconcentration.

➫

In certain cases, nucleic acid fluorescence isobtained for a pH between 1.5 and 3.5. In otherconditions, the pH must be between 3.5 and 5.

5.2.1.4 Action

Orange acridine reacts by intercalation betweenthe two DNA strands or into an RNA loop, bysalt binding or van der Waals forces. The dyeabsorbs at a 520-nm wavelength and the emis-sion is variable as a function of the fresh orpreserved state of the tissue.

(CH3)2N N(CH3)2+

NH

CI


5.2 Staining Methods

119

5.2.1.5 Protocol

❑

Preservation

All fixatives are convenient. It is also possibleto use nonpreserved fresh tissues, smears, or cellcultures.

❑

Reagents

�

Acetic acid 1%

�

Orange acridine 0.1%

�

Phosphate buffer

M

/15, pH 6:

Monopotassium phosphate (Solution 1):

—Monopotassium phosphate

3.068 g

—Distilled water

1000 mL

Disodium phosphate (Solution 2):

—Disodium phosphate

11.88 g

—Distilled water

1000 mL

Buffer:

—Solution 1

87.9 mL

—Solution 2

12.1 mL

�

Calcium chloride

M

/10

❑

Protocol

1. Hydrate.2. Immerse in acetic acid 1%.

6 s

3. Rinse with distilled water.

2

×

3 s

4. Immerse in orange acridine 1%.

3 min

5. Immerse in phosphate buffer

M

/15.

1 min

6. Immerse in calcium chloride.

30 s

7. Mount with phosphate buffer pH 6.

❑

Results

DNA emits a green fluorescence; RNA, a redfluorescence.

➫

As a function of the tissue preparation, thestaining method varies.

➫

For smears:

�

Use ethanol-ether (1:1).

30 min

➫

For paraffin sections:

�

Use a solution mixed with ethanol.

➫

Smears:

�

Ethanol 80%

10 s

�

Ethanol 70%

10 s

�

Ethanol 50%

10 s

➫

Sections: Dehydrate as usual.

➫

Preparation mounting can also be done in amounting medium without fluorescence.

5.2.2 Coriphosphine O Staining

5.2.2.1 Mechanism

Coriphosphine O is an acidic dye belonging tothe acridine family. Under certain conditions,this dye yields results that are comparable withthose given by orange acridine. It is not sensitiveto an increased action of ultraviolet light.This dye gives a green fluorescence to DNA.


Fluorescent Methods

120

Cytoplasmic RNA is copper colored, andnucleus RNA is orange fluorescent (Figure 5.2).

5.2.2.2 Formula

Figure 5.2 Coriphosphine O.

5.2.2.3 Action

Coriphosphine O is an intercalating dye that isfixed between two DNA strands or into RNAloops by salt binding or van der Waals forces.The absorption wavelength is 520 nm.

5.2.2.4 Protocol

❑

Preservation

All classic fixatives are convenient. It is alsopossible to use smears, cell cultures.

❑

Reagents

�

Phosphate buffer

M

/15, pH 7:

Monopotassium phosphate (Solution 1):

—Monopotassium phosphate

3.068 g

—Distilled water

1000 mL

Disodium phosphate (Solution 2)

—Disodium phosphate

11.88 g

—Distilled water

1000 mL

Buffer:

—Solution 1

38.8 mL

—Solution 2

61.2 mL

—Phenol

0.2 mL

�

Coriphosphine O

❑

Protocol

1. Hydrate2. Immerse in phosphate buffer pH

7.5 min

3. Immerse in coriphosphine O.

5 min

4. Immerse in phosphate buffer pH 7.1.

10 s

5. Dry in glycerin.6. Mount into a medium without fluorescence.

❑

Results

DNA is green fluorescent, cytoplasmic RNA iscopper fluorescent, and nucleus RNA is orangefluorescent.

➫

In the original method, Carnoy’s fluid isrecommended for smears and also for samplesthat are to be embedded.

➫

Buffer can with replaced by PBS from acommercial source.

➫

In the original method, the mounting is donein liquid paraffin.

(CH3)2N

CH3,HCI

NH

H

N



121

5.2.3 Propidium Iodide Staining

5.2.3.1 Principle

Propidium iodide is an intercalating fluorescentdye. Its excitation wavelength is 370 and 560 nm.It emits a red fluorescence at 623 nm (Figure 5.3).

➫

Propidium iodide can be used with immu-nofluorescent reactions or with

in situ

hybrid-ization using fluorescent probes.

➫

Ethidium bromide is also an intercalatingfluorescent dye, similar to propidium iodide.It is excited at 370 and 530 nm and emits a redfluorescence at 622 nm.

5.2.3.2 Formula

Figure 5.3 Propidium iodide.

5.2.3.3 Protocol

❑

Preservation

All classic fixatives are convenient. The stainingmethod can be used on sections, smears, or cellcultures.

❑

Reagents

�

Propidium iodide

1 g

�

PBS

100 mL

❑

Protocol

1. Dewax, hydrate.2. Immerse in propidium iodide.

30 min

3. Mount without dehydration with a mount-ing medium without fluorescence.

❑

Results

Nuclei and cytoplasm RNA are red fluorescent.

➫

Operate in the dark.

➫

Preparations can be preserved for severaldays or weeks by storing at –20˚C.

5.2.4 Hoechst 33258 Staining

5.2.4.1 Principle

Hoechst 33258, a fluorescent dye that reacts byintercalating between adenine and thymine, isDNA specific. It is essentially used in flux cytom-etry, and can be used in photonic microscopy to

H2N

(CH2)3 (CH3)3

NH2

N+

N+2 I -


Fluorescent Methods

122

stain nuclei, after visualization with a fluorescentmethod. The excitation wavelength is 360 nm.Fluorescence is emitted at 470 nm (Figure 5.4).

5.2.4.2 Formula

Figure 5.4 Hoechst 33258.

5.2.4.3 DNA visualization by Hoechst 33258

❑

Fixative

All classic fixatives are convenient.❑ Reagents

� Hoechst 33258—Sterile distilled water 50 mL—Hoechst 33258 2.5 mg—Thimerosal 5 mg� Hoechst 33258 dye—Stock solution 1 mL—MacIlvaine buffer pH 5.5 10 mL� MacIlvaine buffer—Citric acid 0.1 M 42 mL

—Disodium phosphate 0.2 M 58 mL

❑ Protocol1. Dewax, hydrate2. Immerse in Hoechst 33258. 15 min3. Rinse with distilled water 2 × 5 min4. Let dry in air5. Mount the slide without dehydration witha glycerin buffer or a mounting mediumwithout fluorescence.

❑ ResultsNuclei are green fluorescent.

➫ Testing the effects of the fixative is recom-mended.➫ Stock solution

➫ Working solution

➫ Citric acid 0.1 M:� Citric acid 2.1 g� Distilled water 100 mL

➫ Disodium phosphate:� Disodium phosphate 3.56 g� Distilled water 100 mL

➫ Working solution➫ In the dark

➫ Preparations can be saved for several daysor weeks at a temperature of –20˚C.

5.2.4.4 Visualization of mycoplasma byHoechst 33258

❑ Fixative

H3C

NN N

N

NH

NH

OH



123

For cell cultures, Carnoy’s fluid is recom-mended, although other fixatives can be used.

1. Use cell culture on lamella.2. Immerse in Carnoy’s fluid. 2 min3. Remove excess fixative.4. Immerse in Carnoy’s fluid. 5 min5. Remove excess fixative.6. Let dry. 37˚C

❑ Reagents� Hoechst 33258—Sterile distilled water 50 mL—Hoechst 33258 2.5 mg—Thimerosal 5 mg� Hoechst 33258 dye—Stock solution 1 mL—MacIlvaine buffer, pH 5.5 10 mL� MacIlvaine buffer—Citric acid 0.1 M 42 mL

—Disodium phosphate 0.2 M 58 mL

❑ Protocol1. Dewax, hydrate2. Immerse in Hoechst 33258. 15 min3. Rinse with distilled water 2 × 5 min4. Let dry in air5. Mount the slide without dehydration witha glycerin buffer or a mounting mediumwithout fluorescence.

❑ ResultsCell nuclei are green fluorescent. Mycoplasmaare visualized as small green points or drops onthe cell, the cell membrane, and the spacebetween the cells.

➫ Stock solution

➫ Working solution

➫ Citric acid 0.1 M� Citric acid 2.1 g� Distilled water 100 mL

➫ Disodium phosphate� Disodium phosphate 3.56 g� Distilled water 100 mL

➫ Working solution➫ In the dark

➫ Preparations can be stored for several daysor weeks at a temperature of –20˚C.

5.2.5 Hoechst 33342 Staining

5.2.5.1 Principle

Hoechst 33342 is a fluorescent dye that interca-lates between adenine and thymine It is DNA spe-cific, the excitation wavelength is 340 nm, and itemits a blue fluorescence at 450 nm (Figure 5.5).


Fluorescent Methods

124

5.2.5.2 Formula

Figure 5.5 Hoechst 33342.

5.2.5.3 Protocol

❑ FixativeAll classic fixatives can be used. The stainingmethod can be used on sections, smears, and cellcultures.❑ Reagents

� Hoechst 33342 10-3 M 1 g� PBS buffer 100 mL

❑ Protocol1. Dewax, hydrate.2. Immerse in Hoechst 33342. 30 min3. Mount the slide without dehydration witha glycerin buffer or a mounting mediumwithout fluorescence.

❑ ResultsCell nuclei are blue fluorescent.

➫ Testing the effects of the fixative is recom-mended.

➫ Operate in the dark.

➫ Preparations can be stored for several daysor weeks at a temperature of –20˚C.

5.2.6 Quinacrine Mustard

5.2.6.1 Principle

The use of quinacrine mustard permits the visu-alization of the X chromosome.

5.2.6.2 Method

❑ Reagents� Quinacrine—Quinacrine 5 g—Distilled water 100 mL� Citric acid—Citric acid 0.1 M 1.92 g—Distilled water 100 mL� Disodium phosphate—Disodium phosphate 0.2 M 2.84 g

CH3

CH3OCH2

N N

N

N

N

N

H

H



125

—Distilled water 100 mL� Phosphate citric acid buffer 0.01 M, pH 5.5—Citric acid 0.1 M 9 mL—Disodium phosphate 0.2 M, pH 5.5 11 mL� Phosphate buffer 0.1 M—Monopotassium phosphate 1.36 g—Distilled water 100 mL� Disodium phosphate 0.1 M—Disodium phosphate 1.42 g—Distilled water 100 mL� Phosphate buffer 0.1 M, pH 7.4—Monopotassium phosphate 0.1 M 8 mL—Disodium phosphate 0.1 M 42 mL—Distilled water 100 mL

❑ FixativeMethod is used on smears preserved with etha-nol 95% or Carnoy’s fluid.❑ Protocol

1. Immerse in ethanol 100%. 3 min2. Immerse in ethanol 95%. 3 min3. Immerse in ethanol 80%. 3 min4. Immerse in ethanol 70%. 3 min5. Immerse in ethanol 50%. 3 min6. Rinse with distilled water. 3 min7. Immerse in quinacrine 0.5%. 5 min8. Rinse with distilled water. 2 × 3 min9. Immerse in citric acid phosphate buffer,pH 5.5. 3 min10. Immerse in phosphate buffer, pH 7.4.

2 × 3 min11. Mount with phosphate buffer 0.1 M, pH 7.4.12. Seal with varnish.

❑ ResultsThe X chromosome is visualized as a green flu-orescent point in male cell nuclei.

5.2.7 DAPI and DIPI

DAPI (4′6-diaminido-2-phenylindol) is an inter-calating fluorescent dye that intercalates itselfbetween adenine and thymine. It is DNA specific,the excitation wavelength is 365 nm, and it emitsa blue fluorescence at 420 nm (Figure 5.6).

➫ These fluorescent dyes have been developedfor flux cytometry, and they are only rarely usedto visualize nucleic acids with a photonic micro-scope. Only a brief description is given here.

Figure 5.6 DAPI.

NH

NH2

NH2

N

NH


Fluorescent Methods

126

DIPI (4′6-diaminido-2-phenylindol) is a fluores-cent dye that intercalates itself between adenineand thymine. It is DNA specific, the excitationwavelength is 340 nm, and it emits a green flu-orescence at 465 nm.


Chapter 6

ObservationPhases


Contents

129

Contents

6.1 Mounting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6.1.1 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6.1.2 Mounting after Dehydration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.1.2.1 Dehydration and Canada Balm Mounting . . . . . . . . . . . . . . . . .6.1.2.2 Dehydration and Mounting by Eukitt . . . . . . . . . . . . . . . . . . . .

6.1.3 Aqueous Medium Mounting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6.1.3.1 Kaiser’s Syrup Mounting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6.1.3.2 Crystalmount Mounting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.1.4 Mounting for Celloidin Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6.1.5 Mounting for Wax Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6.1.6 Mounting for Fluorescent Preparations . . . . . . . . . . . . . . . . . . . . . . . .

6.2 Photonic Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6.2.1 Description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6.2.2 Image Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6.2.3 Different Microscopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.2.3.1 Different Microscope Types. . . . . . . . . . . . . . . . . . . . . . . . . . . .6.2.3.2 Light Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6.2.3.3 Fluorescent Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

131131131131131132132132132133133133133134135135135135


6.1 Mounting

131

6.1 MOUNTING

6.1.1 Principle

After staining, sections must be mounted in apermanent manner so that they may be observed.The mounting mode depends on the sectiontype: embedding with paraffin, celloidin wax, orfrozen sections. It is also dependent both on thestaining method itself, because certain methodsdo not support dehydration, and on the chemicalreaction, because the results of certain his-tochemical reactions cannot be preserved forlong periods.

6.1.2 Mounting after Dehydration

After staining or histochemical reaction, sec-tions must be dehydrated. They then must bemounted with a medium that will prevent themfrom becoming aqueous.

6.1.2.1 Dehydration

and

Canada

balmmounting


2 min


5 to 10 min

3. Immerse in butanol.

5 min

4. Immerse in cyclohexane.

10 min

5. Place a drop of Canada balm on a coverslip.6. Put the coverslip on the sections, avoidingair bubbles.7. Let dry.

24 hat 60˚C

➫

Mounting between the slide and coverslipis a critical step. Be careful to avoid air bub-bles, will interfere with a correct observation.

6.1.2.2 Dehydration and mounting by Eukitt

Eukitt is a synthetic medium that dries quicklyin air.


2 min


5 to 10 min


5 min

4. Immerse in cyclohexane.

10 min

5. Place a drop of medium on a coverslip.

➫

Other synthetic media can be used such asDPX, Permount, HSR, and Clarite, which areavailable from commercial distributors of his-tological products.


Observation Phases

132

6. Put the coverslip on the sections, avoidingair bubbles.7. Let dry.

4 hat RT

➫

It is common for air bubbles to be trappedbetween the slide and coverslip after mounting,which obscures a correct observation.

6.1.3 Aqueous Medium Mounting

6.1.3.1 Kaiser’s syrup mounting

❑

Kaiser’s syrup

�

Gelatin

7 g

�

Distilled water

42 mlat 50˚C

�

Glycerin

50 g

�

Phenol

1 g

❑

Protocol

1. Place a drop of Kaiser’s syrup on a slide.2. Cover with a coverslip.3. Seal with paraffin or nail varnish.4. Let rest.

24 hat 4˚C

6.1.3.2 Crystalmount mounting

This medium allows mounting in hydrosolublemedium without sealing.

1. Place a drop of Crystalmount on the sec-tions.2. Let polymerize.

2 h at 37˚C

3. A coverslip is not necessary.

➫

Kaiser’s syrup must be stored at 60˚C toavoid solidification.

➫

Crystalmount medium has several advan-tages. It is a substance that polymerizes veryquickly without bubble formation. When it ispolymerized, the surface is level, smooth, andhard, and it is not necessary to use a coverslip(but it is possible). Crystalmount also supportsimmersion oil, and can be dissolved with tepidwater without affecting the sections.

6.1.4 Mounting for Celloidin Sections

If the celloidin pellicle has been eliminated(i. e., the sections have been adhered with gel-atin), mounting is done in the classic mannerusing Canada balm or a hydrophobic medium,such as Eukitt, after dehydration. If the celloidinpellicle is still present after staining:

1. Immerse in ethanol 95%.2. Immerse in phenol.3. Immerse in xylene.

➫

Never use ethanol 100% because it dissolvesthe celloidin pellicle.


6.2 Photonic Microscope

133

6.1.5 Mounting for Wax Sections

Wax sections can be directly observed, withoutmounting. They can also be covered directlywith a drop of Canada balm or mountingmedium and a coverslip. After drying, they canbe observed.

6.1.6 Mounting for Fluorescent Preparations

Preparations that are to be observed with a flu-orescent microscope must be mounted with amedium without natural fluorescence. Bufferedglycerin is often used, such as Apathy’s syrupor other synthetic substances, for example, Flu-oprep.

1. Place a drop of mounting medium on theslide.2. Cover with a coverslip.3. Seal the coverslip with paraffin or nail var-nish.4. Let rest.

2 hat 4˚C

6.2 PHOTONIC MICROSCOPE

6.2.1 Description

Photonic microscopes (Figure 6.1) consist of:

�

An optical system with a tube with lenseson the tips. These lenses are the objectiveand the ocular.

�

A system of light situated under the prepa-ration that permits observation of the prep-aration.

�

An apparatus to modify the light intensityand the contrast of the picture obtained.

�

A stage on which the sample is placed.

➫

Several lamp types can be incorporated inthe microscope. Today, halogen lamps areoften used. They provide better light than theincandescent lamps that can still be found oncertain older models of the microscope.

➫

The stage can be operated by a pair ofscrews linked to racks. Two verniers, one onthe horizontal axis and the other on the verticalaxis, are used to mark the part of the tissue thatis of interest.


Observation Phases

134

�

A heavy base.

A camera is generally mounted on the micro-scope.

➫

The stage can also be equipped with anautomatic displacement system.

➫

The base must be heavy for stability, whichpermits observation and photography.

➫

The microscope can be coupled to an auto-matic image analyzer.

1 = Light 2 = Condenser 3 = Stage 4 = Objective lens 5 = Ocular lens 6 = Macrometric screw 7 = Micrometric screw 8 = Tube 9 = Support 10 = Base

Figure 6.1 Photonic microscope

.

6.2.2 Image Formation

The power of a lens depends on its focal length.

The enlargement provided by the objective lenscorresponds to the ratio between the distancefrom two sample points and the distance of thesesame points on the sample image. The focallength of a powerful objective lens is shorterthan that of “weak” objective lens. Resolutionis higher for a powerful objective than a for weakone.To obtain overall magnification, multiply theenlarging powers of the objective lens and theocular lens.The overall magnification of a photographicprint is obtained by multiplying the enlargingpowers of the objective lens, of the ocular lens,and of the photographic system itself.

➫

Focal length is defined as the distancebetween the lens center and the convergentpoint of rays that are parallel to the lens axis(convergence point).

➫

Resolution is the ability to see two neigh-boring objects as distinct entities.

➫

Calculation of the overall magnification of a pho-tographic print is complex. On prints, it is useful toindicate the enlargements with a small scale directlywithin the photograph. This scale will be increasedor reduced in correspondence with enlargement andreduction of the print itself.

1

2

4

3

5

6

7

8

9

10


6.2 Photonic Microscope

135

6.2.3 Different Microscopes

6.2.3.1 Different microscope types

Photonic microscopy uses visible, ultraviolet, or,more rarely, infrared light.

For nucleic acid studies, light microscopes andfluorescent microscopes are currently most oftenused.

➫

Wavelengths of visible light range from 0.4(violet) to 0.7 µm (red). Infrared wavelengthsare lightly larger than 0.7 µm. For ultraviolet,they are shorter than 0.4 µm.

➫

Microscopes with a black background,phase-contrast microscopes, and epipolarizationmicroscopes also exist. They are seldom usedto visualize nucleic acids by classic methods.

6.2.3.2 Light microscope

The light goes across the object, which absorbsa part of the light before the light reaches theeye. The image is more or less contrasted ontoa bright background.Light microscopes are currently used for histo-logical and cytological studies.

6.2.3.3 Fluorescent microscope

In the fluorescent microscope, the light sourceis a mercury vapor lamp. The light spectrumranges from the ultraviolet to the infrared, andthe wavelength is filtered.The microscope can be used with a transmittingfluorescence.

The microscope can be used with an epifluores-cence.

The preparation must be observed as quickly aspossible.

➫

In this case, the light goes across the object,as in the classic microscope. The eye receivesthe fluorescence of the object and the ultra-violet rays.

➫

In this case, the ultraviolet light goes acrossthe objective before reaching the object. Theeye receives only fluorescent light that is emit-ted by the object and not the ultraviolet rays.


Chapter 7

Preparation ofProducts


Contents

139

Contents

7.1 Fixatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.1.1 Preparation of Fixatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7.1.1.1 Alcohol–Formalin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.1.1.2 Baker’s Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.1.1.3 Bouin’s Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.1.1.4 Bouin–Hollande . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.1.1.5 Carnoy’s Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.1.1.6 Flemming’s Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.1.1.7 Formalin–Calcium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.1.1.8 Neutral Formalin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.1.1.9 Salt Formalin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7.1.1.10 Buffered Formalin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.1.1.11 Halmi’s Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.1.1.12 Helly’s Fluid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.1.1.13 Heidenhain’s Susa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.1.1.14 Zenker’s Fluid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7.1.2 Fixation Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2 Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7.2.1 Nuclear Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.1.1 Acetocarmine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.1.2 Azocarmine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.1.3 Borated Carmine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.1.4 Carmalum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.1.5 Groat’s Hematoxylin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.1.6 Hematoxylin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.1.7 Masson’s Hematoxylin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.1.8 Nuclear Fast Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.1.9 Regaud’s Hematoxylin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7.2.2 Background Coloration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.1 Acetic Light Green. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.2 Acidic Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.3 Acidic Fuchsin and Culvert . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.4 Alizarin Acid Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.5 Altmann’s Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.6 Anilin Blue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.7 Calleja’s Picro Indigo Carmine . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.8 Eosin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.9 Eosin–Light Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7.2.2.10 Erythrosin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.11 Erythrosin–G Orange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.12 Fast Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.13 Heidenhain Blue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.14 One-Time Trichroma (Gabe’s Formula). . . . . . . . . . . . . . . . . . .7.2.2.15 One-Time Trichroma (Martoja’s Formula) . . . . . . . . . . . . . . . .7.2.2.16 Paraldehyde Fuchsin (Gabe’s Formula) . . . . . . . . . . . . . . . . . . .

141141141141141141141141142142142142142142142143143143143143143144144144144145145145145145145145146146146146146146147147147147147148148


Preparation of Products

140

7.2.2.17 Phloxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.18 Saffron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.19 Van Gieson’s Picro Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.2.20 Ziehl’s Fuchsin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7.2.3 Histochemical Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.1 H Acid 2% in Veronal Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.2 Alcian Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.3 Ammonium Silver Nitrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.4 Chloramine T . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.5 Coomassie Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.6 Fast Blue B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.7 Gallocyanine Chromic Lac . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.8 Methyl Green . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.9 Methyl Green (Pollister’s Formula) . . . . . . . . . . . . . . . . . . . . . .

7.2.3.10 Methyl Green–Pyronine (First Formula) . . . . . . . . . . . . . . . . . .7.2.3.11 Methyl Green–Pyronine (Second Formula) . . . . . . . . . . . . . . . .7.2.3.12 Naphthol Yellow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.13 Phosphomolybdic Acid – G orange . . . . . . . . . . . . . . . . . . . . . .7.2.3.14 Pyronine (First Formula) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.15 Pyronine (Second Formula) . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.16 Schiff’s Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.17 Silver Methenamine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.18 SO

2

–Azure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.19 Thionin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.2.3.20 Toluidine Blue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7.3 Alcohol Dilutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.3.1 Dilution of Absolute Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.3.2 Dilution of Ethanol 95% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7.3.3 Dilution of Ethanol 90% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

148149149149149149150150150150150150151151151151151151152152152152152153153153153153154


7.1 Fixatives

141

7.1 FIXATIVES

7.1.1 Preparation of Fixatives

7.1.1.1 Alcohol–formalin

�

Absolute ethanol

90 mL

�

Formalin

10 mL

7.1.1.2 Baker’s fluid

�

Neutral formalin

10 mL

�

Anhydrous calcium chloride10% in distilled water

10 mL

�

Distilled water

80 mL

➫

This fixative has long been used for museumcollections.

➫

This fixative is also called formalin – cal-cium fixative.

➫

It is used for the nervous system, lipids,proteins, and amines.

7.1.1.3 Bouin’s fluid

�

Water saturated solution of picric acid

300 mL

�

Formalin

100 mL

�

Acetic acid

20 mL

➫

Bouin’s fluid is a classic fixative that isacidic and cannot be used for visualization ofcertain components (such as mineral compo-nents).

7.1.1.4 Bouin–Hollande

�

Distilled water

100 mL

�

Copper acetate

2.5 mL

�

Formalin

10 mL

�

Acetic acid

1 mL

�

Picric acid

4 g

➫

See

Section 7.1.1.3.

➫

Bouin–Hollande fluid is a classic fixativethat is acidic and cannot be used for visualiza-tion of certain components (such as mineralones).

7.1.1.5 Carnoy’s fluid

�

Absolute ethanol

60 mL

�

Chloroform

30 mL

�

Acetic acid

10 mL

➫

Absolute ethanol can be replaced with meth-anol.

➫

This fixative must be prepared at time ofuse. It is recommended for nucleic acid visu-alization and for study of the nervous system(Nissl bodies).

7.1.1.6 Flemming’s fluid

�

Chromic oxide 1%

75 mL

�

Osmium tetroxide 2%

20 mL

�

Acetic acid

5 mL

➫

This fixative must be prepared at time of use.

➫

It is used for cytological studies and can beused for lipid visualization.



142

7.1.1.7 Formalin–calcium

➫

See

Baker’s fluid, Section 7.1.1.2.

7.1.1.8 Neutral formalin

�

Dilute formalin 39% in distilled water threetimes to obtain formalin 10%. Then neutral-ize with saturated calcium carbonate.

�

Verify with pH paper (pH 7).

➫

Neutral formalin has multiple uses. It can beadvantageously replaced with buffered forma-lin.

7.1.1.9 Salt formalin

�

Neutral formalin

10 mL

�

Physiological serum

10 mL

7.1.1.10 Buffered formalin

➫

Neutral formalin has multiple uses. It canbe advantageously replaced with buffered for-malin.

❑

Fixative

�

Formalin 35%

200 mL

�

Phosphate buffer

1000 mL

❑

Phosphate buffer

�

Solution A

400 mL

�

Solution B

700 mL

❑

Solution A: Disodium phosphate 0.1 M

�

Disodium phosphate

35.817 g

�

Distilled water

1000 mL

❑

Solution B: Monopotassium phosphate, 0.1 M

�

Monopotassium phosphate

13.609 g

�

Distilled water

1000 mL

➫

0.1

M

, pH 7

➫

Na

2

HPO

4

, 12H

2

O, molecular weight:358.17 g

➫

KH

2

PO

4

, molecular weight: 136.09 g

➫

Buffered formalin has multiple uses.

7.1.1.11 Halmi’s fluid

�

Water saturated picric acid

10 mL

�

Heidenhain’s Susa

90 mL

7.1.1.12 Helly’s fluid

�

Zenker’s fluid

100 mL

�

Neutral formalin

5 mL

➫

This fixative is also called Zenker – formalin.

➫

Use stock solution.

➫

This fixative can be used for cytologicalstudies.

7.1.1.13 Heidenhain’s Susa

�

Mercuric chloride

4.5 g

�

Sodium chloride

0.5 g

➫



7.2 Dyes

143

�

Distilled water

80 mL

�

Trichloracetic acid

2 g

�

Acetic acid

4 mL

�

Formalin

20 mL

7.1.1.14 Zenker’s fluid

❑

Stock solution

�

Potassium dichromate

2.5 g

�

Mercuric chloride

5 g

�

Sodium sulfate

1 g

�

Distilled water

100 mL

❑

Working solution

�

Stock solution

�

Acetic acid

5 mL

➫


7.1.2 Fixation Duration

�

Bouin’s fluid

24 to 48 h

�

Carnoy’s fluid

4 h

�

Formalin

Indefinite

➫

Determination of the optimal fixation dura-tion is recommended for each.

7.2 DYES

7.2.1 Nuclear Dyes

7.2.1.1 Acetocarmine

❑

Stock solution:

�

Carmine 1 g� Acetic solution 45% 200 mL� Let boil 5 min

❑ Working solution� Stock solution 50 mL� Acetic solution 45% 50 mL� Ferric chloride

➫ This staining solution is used on a blockbefore sectioning.

➫ With the addition of ferric chloride, chro-mosomes are violet stained.

7.2.1.2 Azocarmine

� Azocarmine (G or B) 0.1 g� Distilled water 200 mL

➫ Staining with G azocarmine is done at 60˚C.Staining with B azocarmine is done at roomtemperature.



144

� Let boil� Let cool� Acetic acid 2 mL

7.2.1.3 Borated carmine

� Carmine 2 g� Sodium tetraborate 4 g� Distilled water 100 mL

at 100˚C� Ethanol 70% 100 mL� Let rest 3 weeks� Filter

7.2.1.4 Carmalum

� Potassium alum 10 g� Distilled water 100 mL� Carminic acid 1 g� Dissolve at high temperature� Let cool� Filter� Salicylic acid 0.2 g� Potassium alum 1 g

➫ Salicylic acid is used to prevent development ofmicroorganisms. It can be replaced with 1 mL for-malin.

7.2.1.5 Groat’s hematoxylin

❑ Solution 1� Iron and ammonium alum 1 g� Distilled water 50 mL� Concentrated sulfuric acid 0.8 mL

❑ Solution 2� Hematoxylin 0.5 g� Ethanol 95% 50 mL1. After dissolution, mix the two solutions.2. Let rest. 1 h3. Filter.

➫ Can be stored for 1 month.

7.2.1.6 Hematoxylin

� Hematoxylin 10 g� Ethanol 95% 100 mL� Let the hematoxylin artificially “age” by adding:� Potassium iodinate 0.2 g� Hematoxylin 1 g


7.2 Dyes

145

7.2.1.7 Masson’s hematoxylin

� Hematein 0.2 g� Potassium alum 5 g� Distilled water 100 mL� Let boil, then cool and filter� Acetic acid 2 mL

7.2.1.8 Nuclear fast red

� Nuclear fast red 0.1 g� Aluminum sulfate 5 g� Distilled water 100 mL� Let boil� Let cool� Filter

➫ Can be stored for several weeks at 4˚C.

7.2.1.9 Regaud’s hematoxylin

� Aged solution of hematoxylin 10 mL� Glycerin 10 mL� Distilled water 80 mL� Place the solution at 37˚C 12 h� Let cool� Filter ➫ Can be stored indefinitely in a flask.

7.2.2 Background Coloration

7.2.2.1 Acetic light green

� Light green 0.1 g� Distilled water 100 mL� Acetic acid 2 mL

➫ For light green 0.5%, use:� Light green 0.5 g� Distilled water 100 mL� Acetic acid 2 mL

➫ Storage is unlimited.

7.2.2.2 Acidic fuchsin

� Acid fuchsin 0.1 g� Distilled water 200 mL� Acetic acid 1 mL ➫ Storage is unlimited.

7.2.2.3 Acidic fuchsin and culvert� Acidic fuchsin 0.1 g� Culvert 0.2 g� Distilled water 300 mL

After dissolution, add:� Acetic acid 0.6 mL ➫ Storage is unlimited.



146

7.2.2.4 Alizarin acid blue

� Alizarin acid blue 0.5 g� Distilled water 100 mL� Aluminum sulfate 10 g� Let boil 5 min� Let cool 24 h� Filter

➫ Storage is unlimited.

7.2.2.5 Altmann’s fuchsin

� Acidic fuchsin 10 g� Old aniline solution at 1.5%, then filtered 100 mL ➫ Storage is possible for 3 months.

7.2.2.6 Aniline blue

� Aniline blue 0.5 g� Distilled water 100 mL� Let boil� Let cool� Filter� Acetic acid 8 mL� Before use, dilute:—Stock solution 100 mL—Distilled water 200 mL

➫ Storage time of the stock solution is unlim-ited.➫ The diluted solution can be stored for sev-eral months.

7.2.2.7 Calleja’s picro indigo carmine

� Indigo carmine 0.4 g� Picric acid–saturated aqueous solution

100 mL� Let rest� Filter ➫ Can be saved for about 6 months.

7.2.2.8 Eosin

� Eosin 1 g� Distilled water 100 mL ➫ Storage time is unlimited.

7.2.2.9 Eosin–light green

� Eosin 1 g� Light green 0.2 g� Phosphotungstic acid 0.5 g� Distilled water 100 mL

After dissolution, add:� Acetic acid 0.5 mL

➫ Storage time is unlimited.


7.2 Dyes

147

7.2.2.10 Erythrosin

� Erythrosin 1 g� Distilled water 100 mL

After dissolution, add:� Acetic acid 1 drop ➫ Storage time is unlimited.

7.2.2.11 Erythrosin–G orange

� Erythrosin 0.2 g� G orange 0.6 g� Distilled water 100 mL� Acetic acid 1 drop

After dissolution, add:� Formalin 1 mL ➫ Storage time is unlimited.

7.2.2.12 Fast green

� Fast green 1 g� Distilled water 100 mL� Acetic acid 0.5 mL ➫ Storage time is unlimited.

7.2.2.13. Heidenhain blue� Aniline blue 0.2 g� G orange 0.5 g� Distilled water 100 mL� Before use, dilute:—Stock solution 100 mL

—Distilled water 200 mL

➫ Storage time of the stock solution is unlim-ited.➫ The diluted solution can be stored for sev-eral months.

7.2.2.14 One-time trichroma (Gabe’s formula)

� S azorubin 0.5 g� Phosphomolybdic acid 0.5 g� Solid green FCF 0.5 g� Distilled water 100 mL� Acetic acid 1 mL� Martius’s yellow at saturation in this mix-

ture

� Let dissolve 2 h� Filter

➫ Solid green can be replaced with fast greenFCF.

➫ Martius’s yellow can be replaced withhydrophilic naphthol yellow. In this case, use:

� Naphthol yellow 0.01 g� Let dissolve 2 h

➫ In all cases, dissolution time can be prolonged.It will become more and more efficacious.➫ Storage time is unlimited.



148

7.2.2.15 One-time trichroma (Martoja’s formula)

� S azorubin 0.5 g

� Phosphomolybdic acid 0.5 g� Solid green FCF 0.1 g� Martius’s yellow at saturation

100 mL� Acetic acid 1 mL� Let rest several hours� Filter

➫ Fast green FCF can also be used.


7.2.2.16 Paraldehyde fuchsin (Gabe’s formula)

❑ Stock solution

� Basic fuchsin 1 g� Boiling water 200 mL� Let boil 1 min� Let cool� Filter� Hydrochloric acid 2 mL� Paraldehyde 2 mL� Place 1 drop of solution on a paper filter;

when the fuchsin red staining disappears, fil-ter the solution.

� Let the precipitate dry, then dissolve in ethanol70%, at saturation (about 150 mL ethanol).

❑ Working solution

� Stock solution 25 mL� Ethanol 70% 75 mL� Acetic acid 1 mL



➫ Storage time is possible for several months.

7.2.2.17 Phloxin

� Phloxin 1 g� Distilled water 100 mL ➫ Storage time is unlimited.


7.2 Dyes

149

7.2.2.18 Saffron

� Gatinais saffron 10 g� Ethanol 100% 250 mL� Let the saffron dry 12 h

at 37˚C� Crush saffron in a mortar� Ethanol 100%� Warm 12 h

at 37˚C

� Filter

➫ It can be useful to let the saffron dry over-night.

➫ It is possible to warm the solution overnightin a well-corked flask to avoid solvent evapora-tion. It is also possible to extract the saffron withethanol 70% using a Soxhlet engine. In thiscase, proceed to the extraction after severalhours.➫ Can be stored for about 6 months.

7.2.2.19 Van Gieson’s picro fuchsin

� Picric acid–saturated aqueous solution 100 mL

� Acidic fuchsin 1% 5 mL ➫ Fuchsin quantity can be 5 to 15 mL for 100mL of water. This depends on the expectedresults and on the nature of the tissue.➫ Can be stored for several months.

7.2.2.20 Ziehl’s fuchsin

❑ Stock solution� Basic fuchsin 1 g� Phenol 5 g� Ethanol 95% 10 mL

Add progressively:� Distilled water 90 mL� Let rest 1 h� Filter

❑ Working solution� Stock solution 30 mL� Distilled water 90 mL

➫ Only the stock solution can be stored forlong periods.

7.2.3 Histochemical Reagents

7.2.3.1 H acid 2% in veronal buffer, pH 9.2

Veronal buffer:� Hydrochloric acid 8.35 g/L 231 mL� Sodium veronal 20.618 g/L 769 mL

Dye:� H acid 2 g� Veronal buffer 100 mL



150

7.2.3.2 Alcian blue

� Alcian blue 1 g� Acetic acid 1 mL� Distilled water 100 mL

➫ This is the preparation method for alcianblue at pH 2. Other preparation modes exist toobtain alcian blue at different pH values byadding acetic or hydrochloric acid. This dye isessentially used to visualize acidic muco-polysaccharides.

➫ Storage is possible for several months.

7.2.3.3 Ammonium silver nitrate

� Ammonium 28% 10 mL� Silver nitrate 80 mL� Continue to pour ammonium silver nitrate

to dissolve the brown precipitate.

� Add distilled water to dissolve opalescentsolution.

7.2.3.4 Chloramine T

� Chloramine T 1 mL� Phosphate buffer pH 7.5 100 mL

7.2.3.5 Coomassie blue

� Coomassie blue 0.2 g� Methanol 46.5 mL� Acetic acid 7 mL� Distilled water 46.5 mL

7.2.3.6 Fast blue B

� Orthodianisidine 0.2 g� Veronal buffer, pH 9.2 100 mL

➫ Fast blue B is also called orthodianisidine.

7.2.3.7 Gallocyanin chromic lac

� Gallocyanin 0.15 g� Chrome alum 5% 100 mL� Let boil 3 min� Let cool 24 h


7.2 Dyes

151

7.2.3.8 Methyl green

Methyl green always contains impurities, whichmust be eliminated with chloroform extraction.

� Methyl green 2 g� Distilled water 100 mL� Chloroform 50 mL

Decant until chloroform remains colorless.

➫ Methyl green contains methyl violet.

➫ Purified methyl green can be stored for along time at 4˚C. Before each new use, extractthe impurities again with chloroform.

7.2.3.9 Methyl green (Pollister’s formula)

� Purified methyl green 1% 25 mL� Ethanol 95% 20 mL� Glycerol 25 mL� Phenol 0.5 g� Distilled water 100 mL ➫ Storage is possible for several months.

7.2.3.10 Methyl green–pyronine (first formula)

� Methyl green 2% 7.5 mL

� Pyronine 2% 12.5 mL� Distilled water 30 mL

7.2.3.11 Methyl green–pyronine (secondformula)

� Methyl green 1% 15 mL� Pyronine 0.25 g� Phenol 0.5 g� Ethanol 95% 2.5 mL� Glycerol 20 mL� Distilled water 85 mL

7.2.3.12 Naphthol yellow

� Saturated solution in ethanolor� Saturated solution in acetic water (1%)

7.2.3.13 Phosphomolybdic acid – G orange

� G orange 2 g� Distilled water 100 mL� Phosphomolybdic acid 1 g

➫ Naphthol yellow is also called Martius’syellow or Mars’s yellow.➫ For salts that are ethanol soluble

➫ For water-soluble salts

➫Phosphomolybdic acid can be replaced byphosphotungstic acid.

➫Storage time is unlimited.



152

7.2.3.14 Pyronine (first formula)

� Aniline 4 mL� Pyronine 0.1 g� Ethanol 40% 100 mL

7.2.3.15 Pyronine (second formula)

� Pyronine 0.2 g� Distilled water 100 mL

7.2.3.16 Schiff’s reagent

� Basic fuchsin 2 g� Distilled water 400 mL at 100˚C� Let cool to 50˚C� Filter� Hydrochloric acid 2 M 10 mL� Let cool to 25˚C� Potassium metabisulfite 4 g� Let rest 12 h

at 4˚C� Mortar-pounded charcoal 1 g� Strongly agitate 2 min� Filter� Hydrochloric acid 2 M 12 mL

➫ Or hydrochloric acid M 20 mL

➫ Or hydrochloric acid M 20 mL

7.2.3.17 Silver methenamine

� Silver nitrate 5% 5 mL� Tetramine hexamethylene 100 mL

� Borate buffer 0.2 M, pH 8 5 mL

� Distilled water 90 mL

Borate buffer:� Solution A: sodium borate 0.2 M—Boric acid 12.404 g—Sodium hydroxide 100 mL —Distilled water 900 mL� Solution B: hydrochloric acid 0.1 M� Buffer—Solution A 59.9 mL—Solution B 44.1 mL

7.2.3.18 SO2–Azure

� Azure I or A 1 g� Hydrochloric acid M 5 mL� Sodium metabisulfite 5% 1 mL� Distilled water 90 mL ➫Can be stored for several weeks. Add several

drops of metabisulfite 10% before each use.


7.3 Alcohol Dilutions

153

7.2.3.19 Thionin

� Thionin 1 mL� Tartaric acid 0.5 g� Distilled water 100 mL

7.2.3.20 Toluidine blue

� Toluidine blue 0.5 g

� Distilled water 100 mL

➫The quantity can be modified depending onthe concentration required.➫ Distilled water can be replaced with buffer.➫ Storage time is unlimited.

7.3 ALCOHOL DILUTIONS

7.3.1 Dilution of Absolute Ethanol

To obtain the alcohol in the left column, add thenumber of milliliters of water in the right columnto 100 mL of absolute ethanol.

To obtain ethanol 95% 6 mLTo obtain ethanol 90% 13 mLTo obtain ethanol 80% 28 mLTo obtain ethanol 70% 48 mLTo obtain ethanol 60% 73 mLTo obtain ethanol 50% 107 mLTo obtain ethanol 40% 158 mLTo obtain ethanol 30% 242 mL

7.3.2 Dilution of Ethanol 95%

To obtain the alcohol in the left column, add thenumber of milliliters of water in the right columnto 100 mL of ethanol 95%.

To obtain ethanol 90% 6 mLTo obtain ethanol 80% 21 mLTo obtain ethanol 70% 39 mLTo obtain ethanol 60% 63 mLTo obtain ethanol 50% 96 mLTo obtain ethanol 40% 144 mLTo obtain ethanol 30% 224 mL



154

7.3.3 Dilution of Ethanol 90%

To obtain the alcohol in the left column, add thenumber of milliliters of water in the right columnto 100 mL of ethanol 90%.

To obtain ethanol 80% 14 mLTo obtain ethanol 70% 31 mLTo obtain ethanol 60% 54 mLTo obtain ethanol 50% 85 mLTo obtain ethanol 40% 131 mLTo obtain ethanol 30% 206 mL


Chapter 8

Protocols


Contents

157

Contents

8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.2 Fixation of Organs for Nucleic Acid Visualization . . . . . . . . . . . . . . . . . . . .

8.2.1 Preparation of Fixatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.2.1.1 Buffered Formalin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.2.1.2 Carnoy’s Fluid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8.2.2 Dissection and Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.2.3 Dehydration and Paraffin Embedding . . . . . . . . . . . . . . . . . . . . . . . . .8.2.4 Epon Embedding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.2.5 Preparation of Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8.2.5.1 Paraffin Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.2.5.2 Plastic Wax Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.2.5.3 Adhering Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8.3 Staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.3.1 Hematoxylin Phloxin Saffron Staining . . . . . . . . . . . . . . . . . . . . . . . .8.3.2 Masson–Goldner’s Trichroma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.3.3 Romeis’s Azan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.3.4 Pappenheim–Unna’s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.3.5 Brachet’s Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8.3.6 Feulgen and Rossenbeck Reaction. . . . . . . . . . . . . . . . . . . . . . . . . . . .8.3.7 Semi-thin Section Stained by PAS and Toluidine Blue . . . . . . . . . . . .8.3.8 Staining of Nucleic Acids with Orange Acridine. . . . . . . . . . . . . . . . .

159159159159159159160160161161162162162162163164164165166166167


8.1 Introduction

159

8.1 INTRODUCTION

This chapter describes several methods currentlyused to prepare tissues and to visualize nucleicacids.

8.2 FIXATION OF ORGANS FOR NUCLEIC ACID VISUALIZATION

8.2.1 Preparation of Fixative

8.2.1.1 Buffered formalin

❑

Fixative

�

Formalin 37.5%

200 ml

�

Phosphate buffer

1000 ml

❑

Phosphate buffer

�

Solution A

400 ml

�

Solution B

700 ml

❑

Solution A: disodium phosphate 0.1 M

�

Disodium phosphate

35.817 g

�

Distilled water

1000 ml

❑

Solution B: monopotassium phosphate, 0.1 M

�

Monopotassium phosphate

13.609 g

�

Distilled water

1000 ml

➫

Classic fixation by buffered formalin is oneof the most often used methods for anato-mopathologic studies.

➫

0.1

M

, pH 7

➫

Na

2

HPO

4

, 12H

2

O, molecular weight:358.17 g

➫

KH

2

PO

4

, molecular weight: 136.09 g

8.2.1.2 Carnoy’s fluid

�

Absolute ethanol

60 ml

�

Chloroform

30 ml

�

Acetic acid

10 ml

➫

This fixative is recommended for nucleic acidvisualization by histochemical methods.

➫

Prepare immediately before use.

8.2.2 Dissection and Fixation

Organs or dissected pieces of organs before fix-ation must be handled with care to preserve theintegrity of tissue and cell for study. Immerse the


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160

tissue in the fixative as quickly as possible.Duration:

�

Formalin

24 h

�

Carnoy’s fluid

4 h

➫

It is possible to store indefinitely piecespreserved with formalin or ethanol 70%.

➫

It is possible to store pieces indefinitely inbutanol.

8.2.3 Dehydration and Paraffin Embedding

❑

Dehydration


4 h


2

×

12 h


2

×

4 h

Organs that are preserved in Carnoy’s fluid aredirectly immersed in butanol.

❑

Clarification


2

×

12 h

❑

Paraffin impregnation

5. Place the tissue in melted paraffin for 4 to12 h depending on the tissue type: 4 h forliver, kidney, spleen, and lung, and 12 h forother tissues. The temperature used is that ofthe melting point of paraffin.

❑

Embedding

6. Embed the impregnated tissue in a paraffinblock that is made with a mold.


➫

Duration of baths in the different alcoholsolutions can be increased: 24 h for each bathin ethanol 95% and 4 h for each in absolute(100%) ethanol. Conversely, in certain cases,these baths can be decreased (only 1 h in eachbath, but at 40˚C).

➫

Duration of the butanol bath can also bemodified. A lengthened duration that can reach24 h or more is useful for embedding. Butanolcan also allow preservation of tissue fragmentsbefore embedding.

➫

Molds can be Leuckart’s bars, embeddingcases, etc.

➫

In the case of a manipulation error, it issometimes useful to embed the pieces again byimmersing the cut block in melted paraffin.When the piece has melted out of its solidparaffin encasement, remake the block.

8.2.4 Epon Embedding

❑

Reagents

�

Epikote 812

�

DDSA

�

MNA

�

DMP30

�

Propylene oxide

❑

Dehydration


10 min


8.2 Fixation of Organs for Nucleic Acid Visualization

161


10 min


2

×

10 min


10 min

5. Immerse in propylene oxide (4˚C).

10 min

❑

Quick dehydration


2

×

10 min


2

×

10 min

3. Immerse in propylene oxide (4˚C).

10 min

❑

Impregnation and embedding medium

�

Epon A—Epikote 812

31 ml

—DDSA

50 ml

�

Epon B—Epikote 812

50 ml

—MNA

44 ml

�

Embedding medium—Epon A

40 ml

—Epon B

60 ml

—DMP 30

1.7 ml

❑

Substitution medium

�

Embedding medium

50 ml

�

Propylene oxide

50 ml

❑

Substitution

1. Immerse in substitution medium.

1 h

❑

Impregnation

at RT

2. Immerse in impregnation medium.

12 h

❑

Embedding

at RT

Embedding is done in molds with differentforms.

3. Add embedding medium.

2 hat 37˚C

4. Add embedding medium.

3 daysat 60˚C

➫

Epon A and Epon B volumes can be modi-fied. For a soft block, increase Epon B propor-tion; for a hard block, increase Epon Aproportion.

➫

Embedding can be done in gelatin capsules,or in special plastic molds.

8.2.5 Preparation of Sections

8.2.5.1 Paraffin Sections

Paraffin blocks must be cut. First, the paraffinaround the object to be sectioned must beremoved, leaving the object enclosed in a trap-ezoidal paraffin block. The lower and uppersides of the block must be parallel.Blocks are placed on the stage of a verticalmicrotome (Minot’s microtome). Sections areusually cut to obtain 4 to 7 µm thickness (sectionthickness is usually given in micrometers).


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162

The block can be positioned on the stage of themicrotome in several ways. Usually, the blockis placed on the stage with a pair of clamps.Section thickness is usually given directly inmicrometers. Sections can be removed directlyfrom the blade with a brush (avoid steel objectssuch as scalpels, forceps, etc.).

8.2.5.2 Plastic wax sections

The block must be cut. The excess plastic allaround the object is removed, leaving the objectenclosed in a pyramidal plastic block. Blocksare arranged on the stage of a microtome witha mandrel or between two clamps. The knife ismade of glass with a special form. A concaveside is directed to the exterior, which allows oneto remove the sections easily before placingthem on a slide. One can also use an ultramicro-tome in semi-thin section mode. The sectionswill be 0.5 to 1 µm thick.Plastic blocks are easy to cut automatically.

➫

In this case, blocks are made in a gelatincapsule.

8.2.5.3 Adhering sections

Use albuminous water to adhere sections to theslides.

➫

See


8.3 STAINING

8.3.1 Hematoxylin Phloxin Saffron Staining

This staining method uses hematoxylin as anuclear dye and phloxin as a cytoplasmic dye.Saffron is collagen specific.

❑

Fixative

All classic fixatives are convenient.

❑

Reagent

�

Hemalum or Groat’s hematoxylin

� Phloxin 0.5% or 1% in distilled water� Saffron obtained by distillation in ethanol

➫ This staining method is used as a standardin pathologic anatomy. It comes from Mas-son’s trichroma.

➫ Nuclear dye: hematoxylin➫ See Chapter 7: Preparation of Products.➫ See Chapter 7: Preparation of Products.


8.3 Staining

163

❑ Protocol1. Dewax, hydrate.2. Immerse in Groat’s hematoxylin. 5 min3. Wash with tap water. 5 min4. Immerse in phloxin. 3 min5. Rinse.6. Immerse in ethanol 95%. 2 min7. Immerse in ethanol 100%. 2 min8. Immerse in saffron. 10 min9. Immerse in ethanol 100%. Briefly

10. Immerse in butanol, cyclohexane.11. Mount.

❑ ResultsNuclei are blue stained; cytoplasm, musclefibers, and red blood cells are red; collagen isyellow.

➫ See Chapter 7: Preparation of Products.

➫ Mount with Canada balm or with a hydro-phobic medium.

8.3.2 Masson–Goldner’s Trichroma

❑ FixativeAvoid fixatives with osmium tetroxide.❑ Reagents

� Groat’s hematoxylin

� Fuchsin culvert� Molybdic G orange� Acetic sulfo green� Acetic water 1%

❑ Protocol1. Dewax, hydrate.2. Immerse in Groat’s hematoxylin. 5 min3. Immerse in tap water to obtain a bluestaining of sections.4. Immerse in fuchsin culvert. 5 min5. Rinse with acetic water.6. Immerse in molybdic G orange. Briefly7. Immerse in acetic sulfo green. 10 min8. Rinse with acetic acid.9. Dehydrate.

10. Mount.❑ ResultsNuclei are black or dark blue. Background isgray, acidophilic cytoplasm is pink, secretionsare red or green stained. Muscles are red andcollagen fibers green.

➫ This staining method provides good visual-ization of chromatin.

➫ Nuclear dye: hematoxylin➫ Groat’s hematoxylin stains nuclei particu-larly precisely. It is possible to see all thedetails of chromatin repartition. See Chapter 7:Preparation of Products.➫ See Chapter 7: Preparation of Products.➫ See Chapter 7: Preparation of Products.➫ See Chapter 7: Preparation of Products.

➫ Groat’s hematoxylin must be exclusivelyused.

➫ It is often necessary to change the aceticwaters after each slide.



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164

8.3.3 Romeis’s Azan

❑ FixativeAll fixatives can be used. However, those withchromium or osmium should be avoided.❑ Reagents

� G or B azocarmine� Aniline blue� Aniline 1% in ethanol 70%� Acetic acid 1% in ethanol 95%

❑ Protocol1. Dewax; collodion, if necessary; hydrate.2. If sections are from tissue preserved with a fixa-tive containing picric acid, the picric acid can beeliminated by 30 min in ethanol/aniline mixture.3. Incubate in G azocarmine. 1 h


at RT4. Rinse in distilled water.5. Differentiate with aniline ethanol until analmost pure nuclear staining is obtained.

6. Add acetic ethanol. 30 s7. Wash with distilled water. 30 s8. Add phosphomolybdic G orange. 5 min9. Wash with distilled water.

10. Add aniline blue. 10 min11. Differentiate blue with ethanol 95%.12. Dehydrate.13. Mount.

❑ ResultsNuclei and certain cytoplasms are red stained; othercytoplasm is yellow or gray. Collagen is bluestained. Secretions can differ as a function of theirnature. Acid mucopolysaccharides are blue stained.

➫ This staining method provides very precisevisualization of chromatin.

➫ Nuclear dye: azocarmine➫ See Chapter 7: Preparation of Products➫ See Chapter 7: Preparation of Products

➫ Picric acid elimination is optional.

➫ Differentiation must be done under micro-scopic control. Caution: Differentiation canbe extremely fast and can provoke elimina-tion of nuclear staining. In this case, the onlything to do is to repeat the staining!➫ Acetic ethanol stops azocarmine differenti-ation. Length of time in the bath can be pro-longed.

➫ Mount with Canada balm or with a hydro-phoboic medium.

8.3.4 Pappenheim–Unna’s

❑ FixativeCarnoy’s fluid is recommended. Acidic fixativesmust be avoided. However, some fixatives, suchas formalin or Bouin’s fluid, can be used.

➫ Practically all classic fixatives are conve-nient, but brief preservation is recommendedto avoid nucleic acid depolymerization. Onlya few hours are necessary.


8.3 Staining

165

❑ Reagents� Methyl green pyronine

❑ Protocol1. Dewax, hydrate.2. Immerse in methyl green

pyronine. 10 min3. Dry slides on a filter paper.4. Dehydrate slides with two quick dips in butanol.5. Immerse in cyclohexane. 10 min6. Mount.

❑ ResultsNuclei DNA and RNAs are purple-blue stained.In cytoplasm, RNAs are pinkish. Acidic muco-polysaccharides can be purple stained.

➫ DNA dye is methyl green.➫ RNA dye is pyronine.➫ See Chapter 7: Preparation of Products.➫ Do not forget to purify methyl green.


8.3.5 Brachet’s Test

❑ FixativeCarnoy’s fluid is recommended. Acidic fixativemust be avoided. However, some fixatives, suchas formalin or Bouin’s fluid, can be used.

❑ Reagents� Crystallized ribonuclease 0.01% in dis-

tilled water� Methyl green pyronine

❑ Protocol1. Prepare three groups of deparaffinedslides, which are neither collodioned norhydrated.2. Treat one group with ribonuclease.

1 hat 37˚C

3. Wash with tap water.4. Treat the second group with distilledwater. 1 h

at 37˚C5. Stain the three groups with methyl greenpyronine.6. Mount.

❑ ResultsA pure green staining of DNA must be observedon the slide treated with ribonuclease.

➫ Practically all classic fixatives are conve-nient, but brief preservation is recommendedto avoid nucleic acid depolymerization. Onlya few hours are necessary.

➫ DNA dye is methyl green.➫ RNA dye is pyronine.➫See Chapter 7: Preparation of Products.

➫ Mount with Canada balm or with an hydro-phobous medium.


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166

8.3.6 Feulgen and Rossenbeck Reaction

❑ FixativeCarnoy’s fluid is recommended, but it is possibleto use numerous other fixatives.

❑ Reagents� Schiff’s reagent� Hydrochloric acid M� Sulfurous water:—Sodium metabisulfite 10%—Metabisulfite 10 ml—Distilled water 190 ml


at 60˚C3. Rinse with tap water. 8 min4. Immerse in Schiff’s reagent. 1 h5. Rinse with tap water. 5 min6. Rinse in sulfurous water. 3 × 1 min7. Rinse with tap water. 5 min8. Dehydrate.9. Mount.

❑ ResultsDNA is red stained.

➫ If another fixative is used, it is necessary todetermine the optimal duration of hydrolysisfor each.

➫ See Chapter 7: Preparation of Products.➫ DNA staining is attained with Schiff’sreagent.

➫ The time given is for tissues preserved withCarnoy’s fluid.


8.3.7 Semi-thin Section Stained by PAS and Toluidine Blue

❑ FixativeAll classic fixatives can be used. Glutaralde-hyde–paraformaldehyde solution is also com-monly used.❑ Reagents

� Periodic acid 1%� Schiff’s reagent� Sulfurous water:—Sodium metabisulfite 1 ml—Distilled water 20 ml—Hydrochloric acid M 1 ml� Toluidine blue (pH 11):—Toluidine blue 2.5 g—Sodium carbonate 0.5 g/L 50 ml—Stir, let boil, filter

➫ DNA is stained with toluidine blue.


8.3 Staining

167

❑ ProtocolSections can be stained before mounting on theslides by carriying them to the different dyevessels with a forceps. Sections can also bemounted on a slide before staining. In this case,use the classic method.

1. Immerse in periodic acid. 15 min2. Rinse with distilled water. 2 × 1 min3. Immerse in Schiff’s reagent. 30 min4. Immerse in sulfurous water. 2 × 2 min5. Rinse with distilled water.6. Immerse in toluidine blue. 1 min7. Rinse with distilled water.8. Mount sections on a slide if staining hasbeen done on sections.9. Eliminate water with filter paper.

10. Let dry in air.11. Mount.

❑ ResultsGlycogen is pink; cytoplasm and nuclei are blueto purple.

➫It is possible to mount with Canada balmwith a hydrophobic medium.➫ Purple staining of nucleic acids and othersubstances is linked to the metachromatic qual-ities of the dye.

8.3.8 Staining of Nucleic Acids with Orange Acridine

❑ PreservationAll the fixatives are convenient. It is also possi-ble to stain nonpreserved fresh tissues, smears,or cell cultures.

❑ Reagents� Acetic acid 1%� Orange acridine 0.1%� Phosphate buffer M/15, pH 6� Calcium chloride M/10

❑ Protocol1. Hydrate.2. Immerse in acetic acid 1%. 6 s3. Rinse with distilled water. 2 × 3 s4. Immerse in orange acridine 1%. 3 min5. Immerse in phosphate buffer M/15. 1 min6. Immerse in calcium chloride. 30 s7. Mount with phosphate buffer, pH 6.

❑ ResultsDNA emits a green fluorescence; RNA a redfluorescence.

➫ For paraffin sections.➫ Use a mixture with ethanol.

➫ Nuclear fluorescent dye: orange acridine.

➫ Preparation mounting can also be done in amounting medium without fluorescence.


Examples of

Staining

Methods

0043Ex_1.fm Page 169 Thursday, October 5, 2000 10:41 AM

Examples of Staining Methods

171

Fig

ure

1

Met

hod

:H

emat

oxyl

in –

phl

oxin

e sa

ffro

n

Tis

sue:

Mou

se e

soph

agus

Pre

para

tion

:

Para

ffin

sect

ion

Fix

ativ

e:

B

ouin

’s fl

uid

Obs

erva

tion

s

:N

czle

i are

dar

k st

aine

d, c

ytop

lasm

is p

ink,

con

nect

ive

tissu

es a

reye

llow

, mus

cles

are

pin

k st

aine

d.

Fig

ure

2

Met

hod

:M

asso

n –

Gol

dner

’ tri

chro

ma

Tis

sue:

You

ng a

mph

ibia

n te

stis

Pre

para

tion

:

Para

f fin

sect

ion

Fix

ativ

e:

B

ouin

’s fl

uid

Obs

erva

tion

s

: N

ucle

i are

dar

k st

aine

d, c

ytop

lasm

is p

ink.



172

˙Fig

ure

3

Met

hod

:M

asso

n’s

tric

hrom

a

Tis

sue:

Mco

use

card

ia

Pre

para

tion

:

Para

ffin

sect

ion

Fix

ativ

e:

Bou

in’s

flui

d

Obs

erva

tion

s

:N

ucle

i are

bro

wn

stai

ned,

cyt

opla

sm is

pin

k, a

nd c

onne

ctiv

e tis

sues

are

gree

n st

aine

d.

Fig

ure

4

Met

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:M

asso

n –

Gol

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’s tr

ichr

oma

Tis

sue:

Mou

se o

vidu

ct

Pre

para

tion

:

Para

f fin

sect

ion

Fix

ativ

e:

Bou

in’ s

flui

d

Obs

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tion

s

:N

ucle

i are

dar

k st

aine

d, c

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lasm

is r

ed, c

onne

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e tis

sue

is g

reen

,m

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es a

re r

ed s

tain

ed.



173

Fig

ure

5

Met

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:M

ouse

eso

phag

us

Tis

sue:

You

ng a

mph

ibia

n te

stis

Pre

para

tion

:

Para

ffin

sect

ion

Fix

ativ

e:

Bou

in’s

flui

d

Obs

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tion

s

:N

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d, c

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, ker

atin

is

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stai

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con

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ive

tissu

e is

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mus

cles

are

yel

low

.

Fig

ure

6

Met

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:H

erla

nt’ s

tetr

achr

oma

Tis

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Mou

se c

ecum

Pre

para

tion

:

Para

f fin

sect

ion

Fix

ativ

e:

Bou

in’ s

flui

d

Obs

erva

tion

s

:N

ucle

i are

red

col

ored

, cyt

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sm is

pin

k or

not

sta

ined

, con

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ive

tissu

e is

blu

e.



174

Fig

ure

7

Met

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:Pr

enan

t’s tr

iple

sta

inin

g

Tis

sue:

Mou

se tr

ache

a

Pre

para

tion

:

Para

ffin

sect

ion

Fix

ativ

e:

Bou

in’s

flui

d

Obs

erva

tion

s

:

N

ucle

i ar

e br

own

stai

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cyt

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sm i

s pi

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e tis

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Fig

ure

8

Met

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:R

omei

s’s

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Tis

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Mou

se e

soph

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Pre

para

tion

:

Para

f fin

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Fix

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Bou

in’ s

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d

Obs

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s

:N

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, cy

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175

Fig

ure

9

Met

Rom

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an

Tis

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Mam

mal

test

is

Pre

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tion

:

Para

ffin

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Fix

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e:

Bou

in’s

flui

d

Obs

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tion

s

:N

ucle

i ar

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d w

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, cy

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is

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,co

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Fig

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10

Met

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:R

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Tis

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Am

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is

Pre

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:

Para

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Fix

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Bou

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Obs

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176

Fig

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11

Met

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Am

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is

Pre

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:

Froz

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Fix

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Buf

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Obs

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Fig

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12

Met

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Pre

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:

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Fix

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Obs

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177

Fig

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13

Met

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Tis

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Pre

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:

Para

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Fix

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Bou

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Obs

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Fig

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14

Met

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MR

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Pre

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Cel

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Fix

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Buf

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Obs

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:N

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.



178

Fig

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15

Met

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Zie

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MR

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Pre

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:

Cel

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Fix

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Buf

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Obs

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Fig

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16

Met

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Para

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179

Fig

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17

Met

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Fig

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18

Met

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:

Cel

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Obs

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s

:N

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180

Fig

ure

19

Met

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:H

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Tis

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MR

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cell

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in w

ith m

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Pre

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:

Cel

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Obs

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s

:N

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are

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Fig

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20

Met

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:H

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3342

Tis

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Cel

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with

apo

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is

Pre

para

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:

Cel

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Obs

erva

tion

s

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Glossary

Glossary Page 181 Thursday, October 5, 2000 10:45 AM

Glossary

183

A________________

Acidic dye

Acidophilic staining

Adenine

Amino acidAuxochrome

B________________

Base

Basic dye

Basophilic staining

C________________

Carbonyl group

Chromogen

ChromophoreChromotrope

Clear bottom microscope

Collodion

Collodioning

D________________

Dehydration

Deoxynucleoside

➫

A salt whose anion is colored and whose cat-ion is not.

➫

Staining that is characterized by the fixationof a colored anion on the tissue or cell element.

➫

A puric base found in nucleic acids, nucleo-sides, and nucleotides. It is linked with thym-ine in DNA and uracil in RNA.

➫

The fundamental element of proteins.

➫

The part of dye fixed upon the tissue or cellto be stained.

➫

A chemical substance that forms a salt whencombined with an acid.

➫

A salt whose cation is colored and whose anionis not.

➫

Staining that is characterized by the fixationof a colored cation on the tissue or cell element.

➫

Chemical groups that are characteristic ofaldehyde and cetone. Their visualization isoften used in histochemistry to reveal nucleicacids or sugars.

➫

Colored chemical substance with a chro-mophore group but without an auxochrome.

➫

Chemical group giving color to a substance.

➫

Tissue or cell substance giving a metachro-matic staining.

➫

Microscope in which the object is imagedon a clear bottom.

➫

Also called celloidin. A form of nitrocellu-lose.

➫

Method consisting of protecting sectionswith a film of collodion.

➫

Successive baths in increasing concentra-tions of solvent.

➫

Molecule constituted by a deoxyribose anda nitrogenous base.


Glossary

184

Deoxynucleotide

DeoxyriboseDuplication

Dye

E________________

Embedding

F________________

Fixation

Fluorescence

Fluorescence microscope

Fluorochrome

G________________

GeneGenotypeGuanine

H________________

HistoneHydrophilic

Hydrophobic

Histochemical method

Histochemistry

➫

Molecule constituted by a deoxyribose, anitrogenous base, and a phosphate.

➫

A sugar belonging to DNA.

➫

Constitution of a new DNA double helixresembling the initial DNA double helix.

➫

Chemical substance that is able to perma-nently stain a tissue or cell component.

➫

Immersion of an organ or tissue in a solidmedium before being cut in sections.

➫

Also called preservation. Fixation consistsof preparing a dead organ to be preserved in astate as close to the living state as possible.

➫

Use of dyes visualized with a fluorescencemicroscope.

➫

Microscope used to observe staining by flu-orescent dyes. It is equipped with an ultravioletlamp.

➫

Fluorescent dye.

➫

Unit of genetic information.

➫

All the genes in an organism.

➫

Puric base found in nucleosides, nucleotides,and nucleic acids.

➫

Protein associated with DNA.

➫

Chemical substance with a great affinity forwater.

➫

Chemical substance with no affinity forwater.

➫

Method of visualization in which conditionsand parameters are controlled.

➫

Part of histology that consists of visualizingtissue and cell components giving a precisechemical composition.


Glossary

185

L________________

Lac

M________________

MicrotomeMetachromasy

Metachromatic dye

Metachromatic form

Mordancy

Mordant

MountingmRNA

N________________

Nuclear dyeNucleic acid

Nucleoside

Nucleotide

O________________

Orthochromatic

P________________

Pentose

Phosphoric groups

➫

A chromogen with a mordant.

➫

Device used to obtain sections.

➫

Quality of certain dyes that stain tissues orcell structures a color that is different from thecolor of the diluted dye solution.

➫

Dye that stains a tissue or cell structure in acolor that is different from the color of diluteddye solution.

➫

Physical form of a dye when it gives ametachromatic staining.

➫

To add a mordant to a chromogene or a tis-sue, permitting staining.

➫

A chemical substance permitting a chromo-gene to be fixed on a tissue.

➫

To protect stained sections with a medium.

➫

Messenger ribonucleic acid. Transmitsgenetic information from nucleus to cyto-plasm.

➫

Dye that stains nuclei (basic dyes).

➫

DNA and RNA. They support and translategenetic information.

➫

Molecule constituted of a sugar and a nitrog-enous base.

➫

Molecule constituted of a sugar, a nitroge-nous base, and a phosphate.

➫

Related to tone values of light and shade ina photograph that correspond to the tones innature.

➫

A class of sugar. Pentoses are part of nucleicacids.

➫

A part of nucleic acids and nucleotides.


Glossary

186

PreservationProgressive staining

Puric base

PurinePyrimidic base

Pyrimidine

R________________

Regressive staining

Replication

RiboseRibosomerRNA

RNase

S________________

Semi-thin section

T________________

Thymine

tRNA

U________________

Uracil

UV

➫

See

Fixation.

➫

Staining in which contact between the dyeand the tissue is stopped when the stain appearsadequate.

➫

Adenine and guanine. They belong tonucleic acids, and they are always linked topyrimidic bases.

➫

A chemical structure.

➫

Cytosine, thymine, and uracil. They belongto nucleic acids and are always linked to puricbases.

➫

A chemical structure.

➫

Staining in which sections are overstainedand the excess dye is then excluded by a dif-ferentiation substance.

➫

Process where genetic information is trans-ferred from DNA to RNA.

➫

A sugar belonging to RNA.

➫

Organelle in which protein synthesis occurs.

➫

Ribosomal ribonucleic acid. RNA belongingto ribosomes

➫

Enzyme used for RNA hydrolysis in controlreactions.

➫

About 0.5- to 1-µm-thick sections.

➫

Pyrimidic base found only in DNA, nucleosides,and nucleotides. It is linked with adenine in DNA.

➫

Transfer ribonucleic acid. RNA that transfersamino acids when protein synthesis occurs.

➫

Pyrimidic base found only in RNA, nucleosides,and nucleotides. It is linked with adenine in RNA.

➫

Ultraviolet light.


Index

0043/index/frame Page 187 Thursday, October 5, 2000 10:46 AM

189

Index

A

Acetic acid fixative, 28Acetic light green, preparation of, 145Acetocarmine, preparation of, 143Acidic fuchsin, preparation of, 145Acidic fuchsin and culvert, preparation of, 145Acidophilic staining, 14Actinomycin method, radioactive, 111Adenine, 5Agar-agar-paraffin, embedding, 38–39Alcian blue, preparation of, 150Alcohol dilutions, 153–154Alcohol-formalin fixative, preparation of, 141Alizarin acid blue, preparation of, 146A-DNA, 7Altmann fuchsin, preparation of, 146Ammonium silver nitrate, preparation of, 150Anaphase, 9Aniline blue, preparation of, 146Auxochrome, 12Azan staining, 63–65

modified, 65–66Azocarmine, preparation of, 143–144Azoic dye staining, 63–65Azure blue staining, 15, 17

B

Bacteria, DNA of, 9Baker fixative, preparation of, 141Bases

complementary, 7detection of,

see

Puric and pyrimidic base detection methods

nitrogenous, 5numbering atoms of, 5

Basophilic staining, 14–15Basophilic staining methods, 92

control staining, 96Gallocyanin method, 92–93Love and Lile method, 98–101Love and Suskind method, 98–101Mann-Dominici staining, 97–98methyl green method, 93–94Pappenheim-Unna method, 95pyronine method, 94ribonuclease Brachet test, 96RNA extraction by hydrochloric acid, 96–97Toluidine blue staining of thin sections, 98

Benson staining method, 108–109

β

-DNA, 7Bolles-Lee fixative, 37Bone, embedding, 43Bone sections

adhesion of, to slide, 51preparation of, 47

Borated carmine, preparation of, 144Borel tubes, 19Bouin fixative

duration of tissue in, 143preparation of, 141

Bouin-Hollande fixative, preparation of, 141Brachet test, in basophilic staining, 96Brachet test staining method, 164–165Buffered formalin fixative, preparation of, 142, 159

C

Calleja picro indigo carmine, preparation of, 146Canada balm mounting, 131Carbonyl groups, staining, 18–19Carmalum

preparation of, 144staining with, 60–61

Carmine, 59borated

preparation of, 144staining with, 59–60

carmalum staining, 60–61preparation of, 59, 143–144

Carnoy fixativeduration of tissue in, 143preparation of, 141, 159

Caspersson spectrophotometric method, for puric and pyrimidic bases, 85

Cell culturesmonolayer, 25–26

preparation of, 54suspension, 26

preparation of, 54Cell cycle, 8–9Celloidin, 13

use in section protection on slides, 52Celloidin embedding, 36–37

with paraffin, 37–38sectioning blocks in, 47

Celloidin sectionsadhesion of, to slide, 49–50mounting, 132


Index

190

Celloidin-paraffin embedding, 37–38sectioning blocks in, 43–46

Celloidin-paraffin sections, adhesion of, to slides, 48–49

Chloramine T, preparation of, 150Chromophore, 12Chromosomes, duplication of, 8–9Clamping with dinitrofluorobenzene, for puric and

pyrimidic bases, 87–88Clamping with performic acid, for puric and

pyrimidic bases, 87Cleveland and Wolfe trichroma staining, 75Cold fixation, 34Collodion, 13,

see also

CelloidinContrast staining, 68

Cleveland and Wolfe trichroma, 75hematoxylin picro indigo carmine, 69–70hematoxylin-eosin, 68–69hematoxylin-phloxin, 69Herlant tetrachroma, 76Masson trichroma, 70–72Masson-Goldner trichroma, 72May-Grünwald Giemsa, for smears, 78–79one-time trichroma, 74–75Pappenheim panoptic, 78paraldehyde fuchsin, 76–77Prenant triple, 72–73Ramon y Cajal trichroma, 74

Control staining, in basophilic histochemical methods, 96

Coomassie blue, preparation of, 150Coomassie blue staining, for proteins, 91Coriphosphine O staining method, 119–120Cresyl blue staining, 17Cryodesiccation, 33Crystalmount medium, 132Cytosine, 5

D

Danielli tetrazoreaction staining methodfor proteins, 90for puric and pyrimidic bases, 86–87

after benzoylation or acetylation, 85–86DAPI staining method, 125Deoxynucleoside, 5Deoxynucleotide, 5Deoxyribose, 5Dinitrofluorobenzene, clamping with, for puric and

pyrimidic bases, 87–88DIPI staining method, 125DNA

in bacteria, 9discovery of, 6

duplication of, 8–9helical structure of, 6in mitochondria, 9in plastids, 9quantitative analysis of, 19, 111size of molecule of, 6stability of, 7structure of, 5–7

variations of, 7viral, 10visualization of, 59,

see also

Fluorescent visualization methods; Histochemical visualization methods

Durcupant embedding, 41Dyes,

see also

Stainingacidic and basic, 12–13chemical groups of, 12definition of, 11fluorescent, 117–118,

see also

Fluorescent dyesmechanism of staining with, 11–12mordants and, 13natural, 12nuclear, 59–68,

see also

Nuclear dyesin staining phenomenon, 11synthetic, 12

E

Embeddingagar-agar and paraffin, 38–39bone, 43celloidin, 36–37celloidin and paraffin, 37–38double, 37–38, 38–39epon, 39–40, 160–161gelatin, 38hard tissue, 41–43paraffin, 34–35, 160–161paraplast, 35–36resin, 39–41

Eosin, preparation of, 146Eosin-light green, preparation of, 146Epon embedding, 39–40, 160–161Erythrosin, preparation of, 147Erythrosin-T orange, preparation of, 147Ethanol fixative, 28Eukitt mounting, 131–132

F

Fast blue B, preparation of, 150Fast green, preparation of, 147Feulgen and Rossenbeck nuclear reaction, 101–102

acidic hydrolysis in, 102


Index

191

alternative methods to, 107Benson method, 108–109Himes and Moriber method, 108Korson method, 109–110silver methenamine method, 109–110thionin-SO

2

method, 107hydrolysis of tissue in, 104–105pentose detection in, 89principle of, 101protocol for, 105–107Schiff reagent in, 102–103tissue preservation/fixation in, 103

Feulgen and Rossenbeck staining method, 166Fixation

chemical, 30–33chemical and physical, 34definition of, 26duration of, 30, 143effects of, 27by formalin vapor, 32holding fluids for tissues in, 34by immersion, 31importance of, 26–27nucleic acid, 30by perfusion, 31–32physical, 33–34thin section, 32–33tolerant and intolerant, 27

Fixative(s)chemical action of, 27coagulant, 28definition of, 28duration of sample in, 30glutaraldehyde/paraformaldehyde, 32–33mixtures of, 29–30noncoagulant, 28–29osmium tetroxide, 33preparation of

alcohol-formalin, 141Baker, 141Bouin, 141Bouin-Hollande, 141buffered formalin, 142Carnoy, 141Flemming, 141formalin-calcium,

see

Baker fixativeHalmi, 142Heidenhain Susa, 142–143Helly, 142neutral formalin, 142salt formalin, 142Zenker, 143

Flemming fixative, preparation of, 141Fluorescent dyes, 117

advantages of, 118Feulgen-Schiff-like fluorescent dyes, 117intercalating, 117pair base-specific, 117

Fluorescent microscope, 135Fluorescent preparations, mounting, 133Fluorescent staining methods, 117

coriphosphine O method, 119–120DAPI, 125DIPI, 125Hoechst 33258 method, 121–123Hoechst 33342 method, 123–124orange acridine method, 118–119propidium iodide method, 121quinacrine mustard method, 124–125

Flux cytometry, 19fluorescent dye use in, 117, 125–126

Focal length, 134Formaldehyde fixative, 28Formalin fixative, 27

buffered, preparation of, 142duration of tissue in, 143neutral, preparation of, 142salt, preparation of, 142vaporized, 32

Formalin-calcium fixative, preparation of,

see

Baker fixative

Freezing-dissolution, 33Frozen sections

adhesion of, to slide, 51preparation of, 47

G

Gallocyanin chromic lac, preparation of, 150Gallocyanin staining method, 92–93Gelatin-paraffin embedding, 38

sectioning blocks in, 43–46Gelatin-paraffin sections, adhesion of, to slide,

48–49Genome, 7–8Glutaraldehyde/paraformaldehyde fixative,

preparation of, 32–33Groat hematoxylin, preparation of, 144Guanine, 5

H

Halmi fixative, preparation of, 142Hard tissue

calcified, 42decalcification of, 42embedding, 43noncalcified, 42types of, 41


Index

192

Hartig-Zacharias staining method, for proteins, 90Heidenhain azan staining, 63–64Heidenhain blue, preparation of, 147Heidenhain Susa fixative, preparation of, 142–143Helly fixative, preparation of, 142Hemalum, 61, 62Hematoxylin

Regaud, preparation of, 145Hematoxylin, preparation of, 144–145Hematoxylin staining

preparations in, 62–63principle of, 61types of, 62

Hematoxylin-eosin staining, 68–69Hematoxylin-phloxin staining, 69Hematoxylin-phloxin-saffron staining method,

162–163Hematoxylin-picro-indigo-carmine staining, 69–70Herlant tetrachroma staining, 76Himes and Moriber staining method, 108Histochemical visualization methods, 85

alternative, 107–110basophilic reactions in, 92

control staining, 96Gallocyanin method, 92–93Love and Lile method, 98–101Love and Suskind method, 98–101Mann-Dominici method, 97–98methyl green method, 93–94Pappenheim-Unna method, 95pyronine method, 94ribonuclease Brachet test, 96RNA extraction by hydrochloric acid, 96–97toluidine blue staining of thin sections, 98

Benson method, 108–109Feulgen and Rossenbeck nuclear reaction in,

101–102hydrolysis of tissue, 104–105protocol for, 105–107Schiff reagent, 102–103tissue preservation/fixation, 103

Himes and Moriber method, 108Korson method, 109–110other, 110pentose detection in

Feulgen and Rossenbeck nuclear reaction, 89

Turchini, et al., method of, 88–89periodic acid-silver diamine method, 110–111protein detection in, 90

Coomassie blue, 91Danielli tetrazoreaction, 90Hartig-Zacharias method, 90T chloramine-Schiff method, 90–91

puric and pyrimidic base detection inCaspersson spectrophotometric method, 85clamping with dinitrofluorobenzene, 87–88clamping with performic acid, 87Danielli tetrazoreaction, 86–87Danielli tetrazoreaction after benzoylation or

acetylation, 85–86quantification of DNA in, 111radioactive actinomycin method, 111reagent preparation for, 149–153,

see also

specific reagent

silver methenamine method, 109–110thionin-SO

2

method, 107Histones, 11

bonding in nucleoproteins, 7Histophotometry, 19Hoechst 33258 staining method, 121–123

mycoplasma detection by, 122–123Hoechst 33342 staining method, 123–124Holding fluids, for fixed samples, 34Hydrochloric acid (2%), in veronal buffer,

preparation of, 149

I

Image formation, microscopic, 134Imaging quantitative analysis, 19Immersion fixation, 31Imprint preparation, 52–53Interphase, 9

K

Kaiser syrup mounting, 132Korson staining method, 109–110

L

Lac(s), 61, 62Leuckart bars, 34–35Light microscope, 135Love and Lile staining method, 98–101Love and Suskind staining method, 98–101Lysochromes, 12

M

Magnification, calculation of, 134Mann-Dominici staining, 15Mann-Dominici staining method, 97–98Masson hematoxylin, preparation of, 145Masson trichroma staining, 70–72Masson-Goldner trichroma staining, 72Masson-Goldner trichroma staining method, 163Mercury chloride fixative, 28


Index

193

Messenger RNA, 10Metachromic staining, 15–17

best dyes for, 17chromotropic substances in, 16spectrometric studies of, 16

Metaphase, 8Methyl green, preparation of, 151

Pollister formula, 151Methyl green staining, 15Methyl green staining method, 93–94Methyl green-pyronine, preparation of

first formula, 151second formula, 151

Microscopefluorescent, 135image formation by, 134light, 135photonic, 133–134types of, 135

Microtome sections, 43–44block preparation for, 44difficulties in preparation of, 44–46

Mitochondria, DNA of, 9Mitosis, 8–9Mordant, 13Mounting sections, 131

after dehydrationwith Canada balm, 131by Eukitt, 131–132

with aqueous mediaCrystalmount medium, 132Kaiser syrup, 132

celloidin sections, 132fluorescent preparations, 133paraffin sections, 133

mRNA, 10Mycoplasma nucleic acid visualization, 118,

122–123

N

Naphthol yellow, preparation of, 151Neutral formalin fixative, preparation of, 142Nitrocellulose, explosiveness of, 13Nuclear dyes, 59

azoic, 63–65, 65–66background coloration preparations for, 145–149,

see also

specific dyecarmine, 59–61for contrast staining, 68–80,

see also

Contrast staining

hematoxylin stain, 61–63,

see also

hematoxylin entries

nuclear fast red, 65

preparation of, 143–145,

see also

specific dyein thin section staining, 67–68

Nuclear fast red, preparation of, 145Nuclear fast red staining, 65Nuclear staining, 59,

see also

Fluorescent visualization methods; Histochemical visualization methods

Nucleic acids, 5–11fixation of, 30viral, 10visualization of, histochemical, 11, 85–111,

see also

Histochemical visualization methodsNucleoproteins, 11

histochemical visualization of, 85Nucleoside, 5, 9Nucleotide, 5, 9

O

One-time trichroma, preparation ofGabe formula, 147–148

One-time trichroma staining, 74–75Orange acridine staining method, 118–119, 167Organs,

see

Tissue entriesOrthodianisidine,

see

Fast blue BOsmium tetroxide fixative, 28

preparation of, 33

P

Pappenheim panoptic staining, 78Pappenheim-Unna staining, 95Pappenheim-Unna staining method, 164–165Paraffin embedding, 34–35, 160–161

sectioning blocks from, 43–46, 161–162Paraffin sections

adhesion of, to slides, 48–49deparaffining, and hydration, 51, 160–161mounting, 133

Paraffin-agar-agar embedding, 38–39Paraffin-celloidin embedding, 37–38

sectioning blocks from, 43–46, 161–162Paraffin-celloidin sections, adhesion of, to slides,

48–49Paraldehyde fuchsin (Gabe formula), preparation of,

148Paraldehyde fuchsin staining, 76–77Paraphenylenediamine, thin section staining with,

67–68Paraplast, 35–36,

see also

Plastic waxPentose(s), numbering carbons of, 5Pentose detection methods

Feulgen and Rossenbeck nuclear reaction, 89Turchini, et al., method of, 88–89


Index

194

Performic acid, clamping with, for puric and pyrimidic bases, 87

Perfusion fixation, 31–32Periodic acid-silver diamine staining method,

110–111Phloxin, preparation of, 148Phosphomolybdic acid-G orange, preparation of,

151Photonic microscope, 133–134

image formation in, 134Picric acid fixative, 28Plastic wax embedding, 35–36

sectioning blocks from, 47, 162Plastic wax sections, adhesion of, to slide, 50Plastids, DNA of, 9Prenant triple staining, 72–73Progressive mode, of staining, 14Prophase, 8Propidium iodide staining method, 121Protein detection methods, 90

Coomassie blue, 91Danielli tetrazoreaction, 90Hartig-Zacharias method, 90T chloramine-Schiff method, 90–91

Puric and pyrimidic base detection methodsCaspersson spectrophotometric method, 85clamping with dinitrofluorobenzene, 87–88clamping with performic acid, 87Danielli tetrazoreaction, 86–87Danielli tetrazoreaction after benzoylation or

acetylation, 85–86Pyronine, preparation of

first formula, 152second formula, 152

Pyronine staining, 15Pyronine staining method, 94

Q

Quantitative analysis, 19, 111Quinacrine mustard staining method, 124–125

R

Radioactive actinomycin method, 111Ramon y Cajal trichroma staining, 74Reductive groups, staining, 17–18Regaud hematoxylin, preparation of, 145Resin embedding, 39–41Resolution, microscopic, 134Ribonuclease Brachet test, in basophilic staining, 96Ribonucleoside, 9Ribonucleotide, 9Ribose, 9–10

Ribosomal RNA, 10RNA, 9–10

extraction of, hydrochloric acid, 96–97types of, 10

Romeis azan staining, 64Romeis azan staining method, 164rRNA, 10

S

Saffron, preparation of, 149Salt formalin fixative, preparation of, 142Samples

cell cultures as, 25–26tissue dissections as, 25types of material used as, 25

Schiff reagent, preparation of, 152Section preparation

adhesion to slide, 48celloidin method, 50gelatin method, 50gelatinized slide method, 49gelatinous water method, 49glycerin-albumin method, 48–49Maximow method, 50water method, 48

block microtoming in, 43–44block cutting prior to, 44celloidin blocks, 47difficulties in, 44–46embedded bone, 47frozen samples, 47paraffin, paraffin-celloidin, paraffin-gelatin

blocks, 43–46, 161–162plastic wax blocks, 47

celloidin protection on slides, 52deparaffining, and hydration, 51fixation of thin sections, 32–33mounting, 131–135,

see also

Mounting sectionsstaining,

see

Fluorescent visualization methods; Histochemical visualization methods; specific staining method

Silver methenamine, preparation of, 152Silver methenamine staining method, 109–110Small nuclear RNA, 10Smear(s)

definition of, 52dry blood, on lamella, 53dry blood, on slide, 53imprint, 52nucleic acid staining methods for, 100–101squash, 53staining, 78–79wet, 53


Index

195

snRNA, 10SO

2

-Azure, preparation of, 152Squash preparation, 53Staining, 11–12,

see also

specific staining methodacidophilic, 14azan, 63–65, 65–66azoic, 63–65azure blue, 15, 17basophilic, 14–15carbonyl group, 18–19Cleveland and Wolfe trichroma, 75contrast, 68–80,

see also

Contrast stainingcresyl blue, 17hematoxylin, 61–63hematoxylin-eosin, 68–69hematoxylin-phloxin, 69hematoxylin-picro-indigo-carmine, 69–70Herlant tetrachroma, 76Mann-Dominici, 15Masson trichroma, 70–72Masson-Goldner trichroma, 72May-Grünwald Giemsa, for smears, 78–79metachromic, 15–17methyl green, 15nuclear, 59,

see also

Nuclear dyesone-time trichroma, 74–75Pappenheim panoptic, 78paraldehyde fuchsin, 76–77Prenant triple, 72–73progressive mode, 14quantitative analysis by, 19Ramon y Cajal trichroma, 74reductive group, 17–18thin section, 66–68thionin, 15, 17toluidine blue, 15, 17types of, 13–14vessels for, 19

T

T chloramine-Schiff staining method, for proteins, 90–91

Telophase, 9Thin sections

fixation of, 32–33staining, 66–68,

see also

Toluidine blue staining

Thionin, preparation of, 153Thionin staining, 15, 17Thionin-SO2 staining method, 107Thymine, 5Tissue preparation

cell culture, 54,

see also

Cell culturescelloidin protection of, on slides, 52deparaffining, and hydration of, 51dissection in, 25, 159–160embedding in, 36–43, 160–161,

see also

Embedding

fixation in, 26–34,

see also

Fixation; Fixative(s)

samples in, 25–26sectioning in, 43–51,

see also

Sectionssmears, 52–2–53,

see also

Smear(s)Toluidine blue, preparation of, 153Toluidine blue staining, 15, 17

of thin sections, 66–67, 98Toluidine blue-PAS staining, of thin sections, 67

method for, 166–167Transfer RNA, 10tRNA, 10Trypsin, use of, 26Turchini et al. staining method, pentose detection by,

88–892-D-ribofuranose, 9

U

Uracil, 10

V

Van Gieson picro fuchsin, preparation of, 149Viral nucleic acids, 10

W

Wax,

see

paraffin entries

Z

Z-DNA, 7Zenker fixative, preparation of, 143Ziehl fuchsin, preparation of, 149


genome visualization by classic methods in light microscopy

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