HISTOLOGICAL

TOOLSPresented to: Presented by:

Dr. Ashutosh Nirola Tejinder Pal Singh

Dr. Madhu Gupta

Dr. Vikram Bali

Dr. Priyanka Sharma

Dr. Kanika Singla

Dr. Priyanka Singla

CONTENTS

INTRODUCTION

MICROSCOPY

BIOPSY

TISSUE CULTURE

STAINING

HISTOCHEMISTRY AND CYTOCHEMISTRY

CELL FRACTIONATION

IMMUNOCYTOCHEMISTRY

HYBRIDIZATION TECHNIQUE

SUMMARY

INTRODUCTION

Histology is the study of the microscopic

anatomy of cells and tissues of plants and animals.

- performed by examining a thin slice (section)

of tissue under a light microscope or electron

microscope. enhanced through the use of

histological stains. Histology is an essential tool

of biology and medicine.

- Histopathology, the microscopic study of diseased

tissue, is an important tool in anatomical

pathology.

- Pathologists, are the personnel who perform

histopathological examination and provide

diagnostic information based on their observations

HISTORYIn 1595, Zacharias Janssen built the first microscope.

Magnified 3-9 times the original size. 2 tubes fit together with

lenses on each side, twisted to view the object focused.

ROBERT HOOK -17 TH

CENTURY- At the age of 27- designed a compoundmicroscope-magnified the object by 30 times.

- Using these scopes he made the greatestcontribution in a book he published entitled‘Micrographia’ - detailed view of all theobjects viewed under the microscope.

.

ANTONY VAN

LEEUWENHOEK.

ANTONY VAN LEEUWENHOEK –

FATHER OF MICROSCOPY

- After reading Hooke’s Micrographia- he learned togrind lenses and created over 500 microscopes( 4 inchlong).

- He preferred simple microscopes unlike Janssen andHooke (compound microscopes - 2 lenses).

- Magnified objects by 200 times.

- Until he discovered bacteria .

- In 1683- described the substance

removed from his own teeth, a little

white matter.

- Under microscope, saw many ‘very

little living animalcules’.

- Later termed as bacteria- the

founder being AV Leeuwenhoek.

SIGNIFICANCE

- Detailed structure of chromosomes bearing genes and themechanism underlying genetic expressions in cells are studied underhistology revealing relationship of histology with genetics.

- Several different types of cells are involved in immunologicalreactions.

- These cells are involved in combating disease causing organismsand describe the cellular basis for the body’s reaction to sensitizingagents like pollens etc.

“Histology plays a great role in immunology”

PREPARATION OF TISSUES FOR

MICROSCOPIC EXAMINATION

- The most common procedure used in the study of tissuesis the preparation of histological sections .

- Under light microscope, tissues are examined by

transillumination, Since tissues are too thick for

transillumination they should be sectioned .

Chemical Fixation with Formaldehyde or Others :

1. are used to preserve tissue from degradation

2. to maintain the structure of the cell and of sub-cellular components such as cell

organelles.

3. Main action of these aldehyde fixatives is to cross-link amino groups in proteins

through the formation of CH2 (methylene) linkage.

Fixing

FIXATIVES

Light microscopy - 10% neutral buffered

formalin (4% formaldehyde in phosphate

buffered saline).

Electron microscopy - glutaraldehyde,

usually as a 2.5% solution in phosphate

buffered saline.

FROZEN SECTION

FIXATION

- Frozen section is a rapid way to fix and mount

histology sections.

- It is done using a refrigeration device called a

cryostat.

- The frozen tissue - sliced using a microtome.

- Frozen slices are mounted on a glass slide ,stained the

same way as other methods.

- To fix tissue for certain stain such as antibody linked

immunofluorescence staining.

- Used to determine if a tumour is malignant when it is

found incidentally during surgery on a patient.

PROCESSING -

DEHYDRATION, CLEARING,

AND INFILTRATION

Aim of Tissue Processing

- Remove water from tissues

- Replace it with a medium that solidifies to allow thinsections to be cut.

1. 5 μm (micrometres; 1000 micrometres = 1 mm) thickfor light microscopy

2. 80-100 nm (nanometre; 1,000,000 nanometres =1 mm) thick for electron microscopy

- Light microscopy - paraffin wax is most

frequently used.

- Immiscible with water, the main constituent of

biological tissue.

- Water must first be removed in the process of

dehydration.

- Samples transferred -baths of progressively

more concentrated ethanol to remove the water.

- Hydrophobic clearing agent (such as xylene) to

remove the alcohol.

- A molten paraffin wax, the infiltration agent,

which replaces the xylene.

- Paraffin wax does not provide a sufficiently hard matrix for cutting very thin

sections for electron microscopy.

- Instead, resins are used.

- Epoxy resins are the most commonly employed embedding media.

- OTHER TYPES OF EMBEDDING MEDIA

Carbowax-It is a water soluble wax. Therefore tissues are directly

transferred to water soluble wax after fixation and washing.

Methacrylate: It is easily miscible with alcohol and gives a clear and hard

block when polymerized.

Agar embedding: It is mainly used in double embedding. Used in FNAC.

Gelatin: Its melting point is less than the melting point of agar. Gelatin may

be used when frozen sections are required on friable and necrotic tissues.

• Samples- passed through bathsof increasing concentration ofethanol, removes waterDehydrated

• Xylene- to remove alcoholClearing

• Molten paraffin wax- replaces xylene.Infiltration

EMBEDDING

- Tissues are ready for external embedding

- Tissues are placed in the moulds with liquid embedding material.(agar, gelatin,

wax.),hardened.

- Formalin-fixed, paraffin-embedded (FFPE) tissues may be stored indefinitely at

room temperature, and nucleic acids (both DNA and RNA) may be recovered from

them decades after fixation, making FFPE tissues an important resource for

historical studies in medicine.

SECTIONING

- Vertical sectioning - perpendicular to the surface

of the tissue- usual method.

- Horizontal sectioning - evaluation of the hair

follicles and pilosebaceous units. Tangential to

horizontal sectioning is done in Mohs surgery.

STAINING

- Staining - contrast to the tissue & highlights particular features of interest.

- Hematoxylin and eosin (H&E stain) is the most commonly used light microscopically stain in histology and histopathology.

- Hematoxylin, a basic dye, stains nuclei blue due to an affinity to nucleic acids in the cell nucleus;

- Eosin, an acidic dye, stains the cytoplasm pink.

- Extracellular structure ,collagen and other fibers.

- In connective tissue stain pink.

ACIDIC DYES BASIC DYES

Eosin Hematoxylin /Methylene blue

Carry net negative charge Carry net positive charge

React/bind with cationic

components of the cell/tissue

With anionic components of

cell/tissue

Less specific (as compared with

basic dyes)

Highly pH specific

Acidophilic / Eosinophilic

(cytoplasmic filaments,

intracellular membranous

components, extracellular fibers)

Basophilic substances ( Po4 of

Nucleic acids, So4 of MPS, CO

proteins)

BASIS OF STAINING

- ELASTIC STAINS -chemically react with elastic fibers in aorta, elastic

cartilage ,and in connective tissue –black/blue.

- RETICULAR STAINS –stain type 3 collagen –blue/black.

- METAL STAINS – stain reticular fibres, golgi apparatus & neurofibrils.

FEULGEN STAIN FOR NUCLEAR PROTEIN

1. Acid hydrolyses or cleaves proteins from deoxyribose of DNA leads

to opening of sugar group & formation of aldehyde.

2. Schiff binds and gives magenta color to aldehyde.

3. Can be useful to quantify amount of DNA ( by using spectrophotometry

of Feulgen stained tissue).

TRICHOME STAINS –these are dyes with multiple

constituents mixed together each dye staining different

structure .MALLORY”S TRICHOME most common.

- NUCLEI –BLACK

- CYTOPLASM,KERATIN,MUSCLE FIBRE –RED/PINK

-CONNECTIVE TISSUE-BLUE

-NISSL’S STAIN –BASIC DYE ,it stains basophillic structures

nuclei ,ribosomes.

STAINS USING CHEMICAL

REACTION

Special stain

PAS positive substances Carbohydrate (glycogen) or

carbohydrate rich molecules,

Basement membrane, reticular fibers

Periodic acid cleaves bond between carbon atoms form aldehyde group

Aldehyde binds with Schiff to produce

magenta or pink color

PAS =PERIODIC ACID SCHIFF

MICROSCOPE

There are two types of microscopes

1. Those utilizing light as a source of their illumination

-light microscope

-Polarizing microscope

-Phase contrast microscope

-Interference microscope

-Dark field microscope

-Confocal microscopy

2-Those utilizing a non visible light source

-Ultraviolet microscope

-Electron microscope

A microscope is an instrument designed to make fine details visible.

The microscope must accomplish three tasks: produce a

magnified image of the specimen (magnification), separate

the details in the image (resolution), and render the details

visible to the eye, camera, or other imaging device

(contrast).

LIGHT MICROSCOPE

we see only two dimensional pictures, orientation of

cut gives different patterns

Ocular lens or Eye piece lens further

amplifies the image and projects it into

viewers retina or onto a screen or

photographic plate

Objective lens enlarges the object and

projects its image in direction of the

ocular lens.

Basic Principle:-

Objective = 40 x

Eyepiece = 10 x

Without magnifying glass

Overall M = 40 x 10 x 1 = 400

PHASE CONTRAST MICROSCOPY

Examine unstained preparations, for fresh specimens, living cells.

Based on the fact that light passing through media with different refractive

index slows down and changes direction.

This forms phase differences between two adjoining regions.

These phase differences by means of special optical system are transformed

into differences of light intensity so that the image becomes visible.

THE BASIC PRINCIPLE TO MAKE PHASE CHANGES VISIBLE IN PHASE

CONTRAST MICROSCOPY IS TO SEPARATE THE ILLUMINATING

BACKGROUND LIGHT FROM THE SPECIMEN SCATTERED LIGHT,

WHICH MAKE UP THE FOREGROUND DETAILS, AND TO

MANIPULATE THESE DIFFERENTLY.

THE RING SHAPED ILLUMINATING LIGHT (GREEN) THAT PASSES THE

CONDENSER ANNULUS IS FOCUSED ON THE SPECIMEN BY THE

CONDENSER. SOME OF THE ILLUMINATING LIGHT IS SCATTERED BY

THE SPECIMEN (YELLOW). THE REMAINING LIGHT IS UNAFFECTED

BY THE SPECIMEN AND FORMS THE BACKGROUND LIGHT (RED).

ADVANTAGES:-

increase contrast,

the background is

dimmed by a gray

filter

DARK FIELD MICROSCOPY

Dark Field Microscopy : special condenser

illuminates specimen with strong oblique light

Uses:

• -In auto radiography

-Study crystals in urine

-Study microbes- slender spirochetes

(Treponema pallidum)

DISADVANTAGES

THE MAIN LIMITATION OF DARK FIELD MICROSCOPY IS THE

LOW LIGHT LEVELS SEEN IN THE FINAL IMAGE. THIS MEANS

THE SAMPLE MUST BE VERY STRONGLY ILLUMINATED,

WHICH CAN CAUSE DAMAGE TO THE SAMPLE.

ADVANTAGES

Dark field microscopy is a very simple yet

effective technique and well suited for uses

involving live and unstained biological samples,

such as a smear from a tissue culture or individual, water-borne, single-celled organisms.

Considering the simplicity of the setup, the

quality of images obtained from this technique is

impressive.

POLARIZING MICROSCOPY

A polarizer is a filter that only allows specific light waves

or vibrations to pass through it and focus them in a single

plane. An analyzer, mainly used as a second polarizer

located above the sample, determines the quantity and

the direction of the light that illuminates a sample. Due to

the use of these filters, the polarized light waves vibrate in

one single direction, instead of the normal ones that

vibrate in random directions.

When studying a specimen the light has to pass through a

polarizer (polarizing filter) and then in some cases through

an analyzer – to increase the quality of image contrast.

This type of microscope differs

from the normal one by using a

polarized light, in which the

light waves vibrate in one

direction. Unlike the ones from

normal light that vibrate in

random directions

CONFOCAL MICROSCOPY

It is an optical imaging technique

used to increase optical resolution

and contrast of a micrograph by

using point illumination and a

spatial pinhole to eliminate out of

focus light in specimens

Uses-it enables the reconstruction

of 3-d images .

ADVANTAGES:-

Contrast and definition are dramatically improved due

to the reduction in background fluorescence

DISADVANTAGES:--

-Harmful nature of high-intensity laser irradiation to living

cells and tissues

The high cost of purchasing

FLOURESCENCE MICROSCOPY

PRINCILPLE-when a certain fluorescent

substances are irradiated by light of a

proper wavelength they emit light with

a longer wavelength. Fluorescent

substances appear as brilliant shiny

particles on a dark background

PARTS-for this method microscope has

a strong ultraviolet light source and

special filters that eliminate ultraviolet

light ,are placed after objective lens to

protect observer’s eye .

Dyes used -acridine orange,auramine (tuberclebacilli),quinacrine(Q banding of chromosomes)

Acridine-DNA complex emits a yellow green light and

RNA Acridine orange emits a reddish orange .

It is thus possible to identify and localize nucleic acidsin cells

ELECTRON MICROSCOPY

Electron Microscope (EM): Electrons are suchsmall particles that, like photons in light, theyact as waves. A beam of electrons passesthrough the specimen, then through a seriesof lenses that magnify the image.Types:Transmission (TEM), scanning (SEM)

Mechanism: similar to LM except that beam ofelectrons replace light source

Recording: photoelectric plate or video detector

Specimen preparation:

Fixation: Glutaraldehyde (cross links with proteins),

Osmium tetroxide (reacts with *phospholipids) makes

cell/tissue electron dense for image enhancement

Other steps are same as routine tissue processing except

Plastic is used for embedding

± Diamond knives are used in microtome ( not metal

knives)

WORKING – electrons are produced by high

temperature heating of a metallic filament(cathode) in a vacuum.The emitted electrons are then submitted

to a potential difference of appx 60-100 kv .

The anode is a metallic plate with a small hole in its

centre.electrons are accelerated from the cathode

to anode .some of these particles pass through the

central opening in the anode forming a constant

stream of electrons.The beam is deflected by

electromagnetic lenses in a way roughly analogous to

optical microscope .The image obtained is further enlarged by one or two projecting lenses and is finally

seen on a flourescent screen on a flourescent screen

or is projected onto photographic plates .

SCANNING ELECTRON MICROSCOPE

It permits pseudo 3-dimentionsal view of the surfaces

of cells tissues and organs

Scanning (SEM)

It differs from TEM that electron beam passes across the surface of spectrum (not thru specimen as in TEM)

Resembles Television

Can see 3D pictures

T.E.M: TRANSMISSION ELECTRON

MICROSCOPE

-In transmission electron microscope (TEM), the source of illumination is a beam of electrons of very short wavelength, emitted from a tungsten filament at the top.

-The whole optical system of the microscope is enclosed in vacuum. Air must be evacuated from the column to create a vacuum so that the collision of electrons with air molecules and hence the scattering of electrons are avoided. Along the column, at specific intervals magnetic coils are placed. Just as the light is focused by the glass lenses in a light microscope, these magnetic coils in the electron microscope focus the electron beam.

-The specimen stained with an electron dense material and is placed in the vacuum. The electron beams are passes through the specimen and scattered by the internal structures.

The beam of electron that has been partially

transmitted through the very thin specimen

carries information about the structure of the

specimen. The spatial variation in this information

(the "image") is then magnified by a series of

magnetic lenses until it is recorded by hitting a

fluorescent screen, photographic plate, or light

sensitive sensor like CCD (charge-coupled device) camera. The image detected by the

CCD may be displayed in real time on a monitor

or computer.

Atomic Force M: most powerful tool tostudy surface topography

Non – optical M: works like finger tip

Has highest resolution power – 50 pm

No need to put specimen in vaccum

The tip is

attached to the free end of a cantilever and is

brought very close to a surface. Attractive or repulsive

forces resulting from interactions between the tip and

the surface will cause a positive or negative bending

of the cantilever.

The bending is detected by means of a laser beam,

which is reflected from the back side of the cantilever.

The AFM

measures the forces

acting between a fine

tip and a sample.

EXAMINING LIVING TISSUES

(CULTURE MEDIA)

Prolonged study of living tissues can be achievedby culturing them in solutions that containnecessary nutrients to keep them alive.

Cells are treated prior with trypsin-isolated-cultivated insuspension - spread out on a glasssurface to which they adhere spontaneously as asingle layer of cells.

Culture medium should be changed frequentlysince nutrients become depleted and toxicproducts of metabolism accumulate. Rigorousaseptic techniques are necessary during processof cell cultivation in order to avoid contaminationof culture media

BIOPSY

BIOPSY – it is a controlled and deliberate

removal of tissues from a living organism for

the purpose of microscopic examination.

PURPOSE :

1. Make a speedy, definitive, accurate diagnosis.

2. To aid in determining the prognosis of the lesion.

3. Aids in deciding the best treatment possible.

Several methods of biopsy:

1. Surgical excision by scalpel.

2. Surgical removal by cautery or a high frequency cutting knife.

3. Laser.

4. Removal by biopsy forceps or biopsy punch.

5. Aspiration through a needle with a large lumen.

6. EXFOLIATIVE CYTOLOGY TECHNIQUE

CLASSIFICATION

-Excisional biopsy

- Incisional

- Wedge

o Modification of incisional biopsy

Punch biopsy

Curettage biopsy

Drill biopsy

-Needle biopsy

Aspiration needle biopsy

Fine needle aspiration biopsy

Large needle aspiration biopsy

Core needle aspiration biopsy

Miscellaneous

o Exfoliative cytology

EXCISIONAL BIOPSY

It is referred to as total excision of small lesion (less

than 1cm). 2-3mm normal tissue surrounding the

lesion is also excised.

METHOD

- Give LA which should not becloser than 2cm from the site.

- Stabilize the lesion via thesuture.

- Incise mucosa around the baseof the lesion in an ellipticalshape.

- Place specimen immediately ina fixative.

- Close the wound using suture.

INCISIONAL BIOPSY:

-Some lesions are too large to excise initially without having established diagnosis or are of such a nature that excision would be inadvisable in such instances a small section is removed for examination called incisional or diagnostic biopsy.

-It samples only a particular or representative part of the

lesion

-Lesion is larger than 1cm in diameter

- Lesion should be incisedin wedge fashion.

- Multiple biopsies arepreferred over solitarybiopsy.

- Area selected mustshows complete tissuechanges.

PRINCIPLES OF INCISIONAL BIOPSY

- Tissue specimen - taken from the edge of the lesion along with normal tissue .

Necrotic tissue should be avoided.

Biopsy - deep and narrow biopsy rather than a broad and shallow one, because superficial changes may be different from those deeper in the tissue.

PUNCH BIOPSY

It is considered as a primary technique to

obtain diagnostic full thickness of lesion .

CURETTAGE BIOPSY-

A curette is a surgical instrument with a curved spoon

like tip and a long handle designed for scraping out

body cavities for tissue diagnosis or therapeutic

purposes. The samples produced are usually soft

tissue but may include bone fragments as well.

- The curettage is usually diagnostic for pathological

purposes, but can also be therapeutic in treating the

condition.

TECHNIQUE

Most common method - Fountain Pen Technique

The curette is held between the thumb, index

and middle finger

The skin is stretched with finger of other hand

Other common method - Potato Peeler

Technique

Handle of curette is held in the distal inter-digital

fold of the index finger supported by other fingersof curetting hand

Thumb provides a stable base

CELL ASPIRATE

-Aspiration biopsy is the use of a needle and

syringe to penetrate a lesion for aspiration of its

content

It is relatively painless

Use of local analgesia optional

Capable of producing immediate results

TECHNIQUE

A 18-gauge needle is connected to a 5 or 10 ml syringe

The needle is inserted into the lesion

Tip of needle may have to be repeatedly repositioned to

locate a fluid centre

Fluid is withdrawn through the syringe

If an aspirate of cells is obtained using fine needles (21-

25G) the technique is called 'fine needle aspiration

cytology (FNAC)' or 'fine needle aspiration (FNA)

If a core of tissue is produced using larger bore needles

(14-18G), the procedure is best referred to as 'fine needle

cutting biopsy (FNCB) or trucut biopsy'

VARIOUS ASPIRATES

EXFOLIATIVE CYTOLOGY

It was introduced by Papanicoloau in the year 1943

Oral exfoliative cytology is still controversial, particularly

in premalignant conditions

Normal oral squamous epithelium continuously sheds

the most superficial cells

If the area is affected by malignant or other disease,

the deeper cells lose their cohesiveness and are

exfoliated at the same time as the superficial cells

Exfoliative cytology has

not been a very

diagnostic or useful

screening method for oral

cancer because

hyperkeratosis and keratin

itself interfere with cell

obtainment and a greater

proportion of diagnostic

cells are below the surface

(most at the basement

membrane level)

CYTOLOGICAL SMEARS FALL UNDER

FOLLOWING CATEGORIES

Class 1 (normal) indicates that only normal cells were

observed

Class 2 (atypical) indicates presence of minor atypical

but no evidence of malignant change

Class 3 – this is an in between cytology that separates

cancer from non cancer diagnosis .biopsy is

recommended

Class 4 (suggestive of cancer) a few cells with

malignant characteristics or many cells with borderline

characteristics .biopsy is mandatory

Class 5 (+ve for cancer) cells that are obviously

malignant .biopsy mandatory

HISTOCHEMISTRY AND CYTOCHEMISTRY

• Are used to indicate methods for localization different

substances in tissue sections

• They are based on specific chemical reactions or on

high affinity interactions between macromolecules

• Both methods usually produce insoluble coloured or

electron dense compounds that enable the

localization of specific substances by means of light or

electron microscopy

NUCLEIC ACIDS-DNA can be identified and quantified in

cell nuclei using feulgen reaction which produces a red

color in presence of dna

PROTEINS –specific enzymes such as RNAase

,DNAase,collagenase and elastase that digest cell and

tissue components are used extensively

POLYSACCRIDES and OLIGOSACCRIDES –glycogen

which can be demonstrated by PAS .

GLYCOSAMINOGLYCANS react strongly with ALCIAN

BLUE DYE

Neutral glycoproteins react with PAS but are not

digested by glycogenolytic enzymes .

REACTIONS FREQUENTLY USED IN

LABORATORY DIAGNOSIS

PAS-AMYLASE REACTION and alcian blue reaction –

glycogen and glycosaminoglycans

ENZYMES –acid phosphatase –THE GOMORI METHOD –

consist of incubating formalin fixed tissue sections in a

solution containing sodium glycerophosphate and

lead nitrate buffered to ph.5. The enzymes hydrolyses

the glycerophosphate liberating phosphate ions that

react with lead nitrate to produce an insoluble

,electron dispersing , colourless ppt of lead phosphate

at the site of enzymatic activity

In a second step the preparation is immersed in a solution

of ammonium sulfide that reacts

With lead phosphate to produce a blak ppt of lead sulphite

.this method permits the localization of this enzyme’s activity

and is frequently used to demonstrate lysosomes

,cytoplasmic organelles that contain acid phosphates .

DEHYDROGENASES-these enzyme remove hydrogen from

one substrate and trasfer hydrogen from one substance

and transfer it to an other .the enzyme transports

hydrogen from the substrate to the tetrazole and

reduces it to an intensely coloured insoluble compound

FORMAZAN which ppt at the site of enzyme active

example is succinate dehydrogenase .

Peroxidase –a section of adequately fixed tissue are

incubated in a solution containing hydrogen peroxide

and 3,3’diaminoazobenzidine ,it is oxidised in presence

of peroxidase resulting in black ppt.

IMMUNOCYTOCHEMISTRY

It is based on coupling of immunoglobulin's to substances

that render them visible in microscope without causing a

loss of antibody’s biological activity

METHODS OF LABELLING ANTIBODIES

1.COUPLING WITH A FLOURESCENT ANTIBODIES

2.COUPLING WITH AN ENZYME-Most common enzyme used

is peroxidase

3.COUPLING WITH A COLOURED ELECTRON SCATTERING

COMPOUND e.g gold particle

There are three methods –

Direct, Indirect AND Indirect Method With Signal

Amplification

Direct method –The detection of the complex is based

upon the molecule conjugated to the antibody.

Advantages of direct detection include the ease of use

for multicolor staining and the elimination of concerns

regarding non-specific binding of the secondary antibody.

INDIRECT METHOD indirect detection methods generally have

a higher level of sensitivity and generate a more intense

signal. The signal is amplified using the indirect method

because of the potential for at least two labeled secondary

antibodies to bind to each primary antibody..

Further amplification of the signal can be achieved by taking

advantage of the strong affinity of avidin and streptavidin to

bind biotin. Avidin is a glycoprotein in egg white that

combines stoichiometrically with biotin. Streptavidin is purified

from the bacterium Streptomyces avidinii, is not glycosylated,

and exhibits lower non-specific binding than avidin. Both

proteins bind four biotins per molecule. If a biotinylated

secondary antibody is employed, the signal can be

significantly amplified by subsequent incubation with an

avidin-biotin complex (ABC Method), or labeled streptavidin-

biotin (LSAB Method). Streptavidin may be conjugated to a

detection enzyme

INDIRECT METHOD WITH SIGNAL AMPLIFICATION

HYBRIDIZATION TECHNIQUE

Working of cell is imp to understand and this requires that

technique that permit analysis of molecules involved in

process of information flow from DNA to protein

SOUTHERN ANALYSIS –characterizes and quantifies the

presence of DNA of a specific gene in the presence of all

other genes in a eukaryotic organisms

NORTHERN ANALYSIS –based on high affinity between

complementary sequence of nucleic acids. Identifies and

quantifies specific messenger RNA transcripts in the

presence of all RNA transcripts expressed with in a single

cell type

Western Analysis- detects a single protein

species from among all other proteins expressed

in a single cell or tissue. It is based on high

affinity and specificity between antibodies and

antigens

NUCLEIC ACID PROBES

A probe is a nucleic acid molecule (single-stranded DNA

or RNA) with a strong affinity with a specific target (DNA

or RNA sequence).

DNA PROBES- use segments of single stranded nucleic acid

labelled with an enzyme or radioisotope that is able to

hybridize to the complementry nucleic acid sequence and

thus detects the presence of particular microorganisms

HYBRIDISATION refers to pairing of complementry DNA

strands to produce a double stranded nucleic acid

-More specific oligonucleotide

probes complementary to variable

regions of 16s rRNA

Bacterial genes have been

identified .these bacterial 16s rRNA

genes contain both regions shared

by different bacteria and short

streches of variable regions shared

by specific organisms of same

species or genes .

LECTIN HISTOCHEMISTRY

Lectins are proteins derived mainly from plant seeds thatbind to cell surface carbohydrates with high affinity andspecificity

Different lectins bind to specific sequences of sugarresidues.

They bind to cell surface glycoproteins, proteoglycansand glycolipids and are widely used to characterizemembrane molecules containing specific sequences ofsugar residues.

Lectins are usually labeled with peroxidase to makepossible their identification.

SUMMARY

The small size of cells and matrix components

make histology dependent on use of tools

advances in chemical, biology, physiology,

immunology and pathology and interactions

among these contribute to better knowledge of

tissue biology

Familiarity with the tools and methods of any

branch of science is essential for a proper

understanding of the subject. It was a review of

some common tools used in histology

REFERENCES

Basic Histology (9th Edition) Lange

Shafer’s Oral Pathology (5th Edition)

Orban’s Oral Histology (13th edition)

Dye, Stains & special probes in histology (Wolf D, HulmannN.D)

Cell Biology Lab Manual ( Dr. William H. Heidcamp)

Immunihistochemistry (Eldem Sadikoglou)

Molecular Hybridization Technique Of Nucleic Acid: ISSN1843-6099

Oral Histology (Antonio Nancy 6th Edition)

Microscope : Basic & Beyond (Mortimer Abramowitz Vol.1)

Textbook of Human Histology (Dr. Veena Bharioke)

A Book Of Microbiology –Ananthnarayan

THANK

YOU