basic staining techniques lecture 1 1. objectives: at the completion of this section the student...
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BASIC STAINING TECHNIQUES
LECTURE 1
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OBJECTIVES: At the completion of this section the student will be able to:
Know the steps involved in tissue processing and discuss the different types of microscopy.
lnterprete the cytological significance of differential staining produced by hematoxylin and eosin (H&E).
Recognize and identify cells in microscopic tissue preparations.
Understand that morphology reflects the function of cells.
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Methods
• Histochemistry• Cytochemistry• Immunocytochemistry• Organ & tissue culture• Differential centrifugation• Specialized microscopic techniques
Histology
•is the study of tissue sectioned as a thin slice, using a microtome.
•It can be described as microscopic anatomy.
•Histology is an essential tool of biology.
• Histopathology, the microscopic study of diseased tissue, is an important tool of anatomical pathology since accurate diagnosis of cancer and other diseases usually requires histopathological examination of samples.
• Histochemistry refers to the science of using chemical reactions between laboratory chemicals and components within tissue.
• antibodies are used to specifically visualise proteins, carbohydrates and lipids: this is called immunohistochemistry.
Antibodies are proteins of the globulin group(immunoglobulins) that appear in plasma and tissue fluids after antigen injection,their production enables the organism to eliminate certain proteins and other foreign matter not recognized by self.
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Histochemistry and CytochemistryHistochemistry and Cytochemistry
• Tissue Preparation• Tissue Preparation
Methods for the observation of - living cells- dead cells
Methods for the observation of - living cells- dead cells
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Living CellsLiving Cells
Cells from tissue cultures and unicellular organisms observed with a phase-contrast microscope
Cells from tissue cultures and unicellular organisms observed with a phase-contrast microscope
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Dead cells
Sample sources
Biopsy
Blood samples
Autopsy
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Stages of the Histology TechniqueStages of the Histology Technique
Light microscopyLight microscopy Sampling
Fixation
Dehydration
Clearing
Infiltration (impregnation)
Sectioning
Staining
Dehydration
Clearing
Mounting
Sampling
Fixation
Dehydration
Clearing
Infiltration (impregnation)
Sectioning
Staining
Dehydration
Clearing
Mounting
Embedding
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Sampling
Aim – is to provide a representative specimen
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FixationFixation
Some organs needs fixation even before the macroscopic preparation takes place
Some organs needs fixation even before the macroscopic preparation takes place
10 % formaldehyde is the cheapest and easiest to keep fixative solution
10 % formaldehyde is the cheapest and easiest to keep fixative solution
Aim – to preserve the cells with the “least alteration” possible from the living state
Use of substances that coagulate the protoplasm
Simple – formaldehyde, alcohol, acetic acid, osmic acid, picric acid
Complex – Bouin’s fluid, Zenker’s fluid, special mixtures
Aim – to preserve the cells with the “least alteration” possible from the living state
Use of substances that coagulate the protoplasm
Simple – formaldehyde, alcohol, acetic acid, osmic acid, picric acid
Complex – Bouin’s fluid, Zenker’s fluid, special mixtures
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Dehydration
Aim – to remove water from the sample to allow paraffin impregnation
Done by passing tissue sample through gradually increasing concentrations of alcohol
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ClearingClearing
The name of the process come from the clear appearance the sample gets
The name of the process come from the clear appearance the sample gets
Benzene, cedarwood oil, chloroform and others can also be used
Benzene, cedarwood oil, chloroform and others can also be used
Aim – to take the alcohol out of the sample to allow paraffin to impregnate the tissue
The tissue is immersed in xylene (xylol) that is miscible both in the dehydrating and the embedding agent
Aim – to take the alcohol out of the sample to allow paraffin to impregnate the tissue
The tissue is immersed in xylene (xylol) that is miscible both in the dehydrating and the embedding agent
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InfiltrationInfiltration
The embedded specimen in the plastic frame can be (and should be) safely conserved for many years
The embedded specimen in the plastic frame can be (and should be) safely conserved for many years
It is very important to properly label each one of the plastic frames
It is very important to properly label each one of the plastic frames
Aim – to incrust the sample inside a material that allow easy cutting
The sample goes inside a bath of warm paraffin
The tissue embedded in paraffin is put in a plastic frame that can be used later in the microtome
Aim – to incrust the sample inside a material that allow easy cutting
The sample goes inside a bath of warm paraffin
The tissue embedded in paraffin is put in a plastic frame that can be used later in the microtome
• During this 12 to 16 hour process, paraffin wax will replace the water: soft, moist tissues are turned into a hard paraffin block, which is then placed in a mould containing more molten wax (embedded) and allowed to cool and harden.harden.
• Embedding can also be accomplished using frozen, non-fixed tissue in a freezing medium.
• This freezing medium is liquid at room temperature but when cooled will solidify.
Microtome
• A microtome is a mechanical instrument used to cut biological specimens into very thin segments for microscopic examination.
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SectioningSectioning
Aim – to cut the sample in slices thin enough to be useful
Sections of the tissue are cut with the aid of the microtome
The thickness, between 3 and 10 µm, is selected depending the stain that is going to be used
The section is transferred to a glass slide
Aim – to cut the sample in slices thin enough to be useful
Sections of the tissue are cut with the aid of the microtome
The thickness, between 3 and 10 µm, is selected depending the stain that is going to be used
The section is transferred to a glass slide
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SectioningSectioning
Tissue block is cut into 1~20μm thick sections by microtome, and laid out on the surface of warm water and are then adhered to gel-coated slides.
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StainingStaining
Aim – to add color to the structures of the sample in order to differentiate them
There are several phases of staining
Every stain has a different procedure (phases, time, extra procedures, etc.)
Special stains color different structures
Aim – to add color to the structures of the sample in order to differentiate them
There are several phases of staining
Every stain has a different procedure (phases, time, extra procedures, etc.)
Special stains color different structures
Most common, easiest to use & cheapest stain is hematoxylin and eosin (H and E)
Most common, easiest to use & cheapest stain is hematoxylin and eosin (H and E)
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Dehydration and clearingDehydration and clearing
Before After
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MountingMounting
Aim – to put the final sample in a medium where it is protected and can also be observed under the microscope
The sample is dehydrated again
A drop of mounting agent with a similar refractive index to that of glass is placed on the glass slide, and covered with a glass coverslip is added
Aim – to put the final sample in a medium where it is protected and can also be observed under the microscope
The sample is dehydrated again
A drop of mounting agent with a similar refractive index to that of glass is placed on the glass slide, and covered with a glass coverslip is added
Mounting medium - DPX - Canada Balsam diluted in xylene (xylol)
Mounting medium - DPX - Canada Balsam diluted in xylene (xylol)
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Freeze Drying Method (Important during surgical biopsy diagnosis)
Freeze Drying Method (Important during surgical biopsy diagnosis)
The quality of the section by this method is poor, and a regular histological technique should followed at the end of surgery.The quality of the section by this method is poor, and a regular histological technique should followed at the end of surgery.
• Aim – to quickly prepare a glass slide for rapid study of specimens during a surgical procedure
• Steps involved in specimen preparation: – Freeze the tissue– Dehydrate the sample under a vacuum – Embed the dehydrated sample
***immersion of tissues in xylene dissolves the tissue lipids, which is undesirable effect when these compounds are to be studied.
To avoid loss of lipids, a freezing microtome has been devised in which the tissues are hardened at low temperatures to provide rigidity necessary to permit sectioning.The freezing microtome is a more elaborate and
efficient successor of the cryostat.***
Cell Smears
• Cell smears are a form of histological preparation that does not require sectioning. Smears can be made for example of the blood or bone marrow. Smears are also common for swabs or scrapings of epithelial cells (e.g. from the oral cavity, cervix uteri).
MATERIALS
•- sterilized lancet or needle- 20 clean microscope slides and coverslips- Canada balsam or other medium for permanent preparations- 95% ethyl or methyl alcohol- distilled water- Giemsa stain- low containers (you can make them with aluminum sheet also)- microscope which magnifies 200 times at least
• TAKING THE BLOOD
• MAKING THE SMEAR
• FIXING
• STAINING
• CHECKING
• COVER-SLIPPING
• OBSERVATION
MAKING THE SMEAR
FIXING
• fixing technique consists of dipping the smear in a vessel containing 95% ethyl or methyl alcohol for 3-5 minutes.
STAINING
• To be able to observe and recognize the different kinds of leukocyte, you must stain them. For this purpose, normally Giemsa stain is used.
• It is a mixture of stains, based on methylene blue and eosin
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Electron Microscopy
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Stages of the Histology TechniqueStages of the Histology TechniqueElectron microscopyElectron microscopy
• Small samples (1 mmSmall samples (1 mm3 3 or less)or less)
• Tissue must be freshTissue must be fresh
• Double fixation (Double fixation (glutaraldehyde for proteins plus osmium glutaraldehyde for proteins plus osmium tetroxide for lipids [osmium also stains])tetroxide for lipids [osmium also stains])
• Quick dehydration and clearingQuick dehydration and clearing
• Embedding in plastic resins Embedding in plastic resins (Epon or Araldite)(Epon or Araldite)
• Cut with ultramicrotome (Cut with ultramicrotome (glass or diamond knifesglass or diamond knifes) produces 30 ) produces 30 – 50 nm thick specimens– 50 nm thick specimens
• Mounting in copper gridsMounting in copper grids
• Stain with heavy metals
• Small samples (1 mmSmall samples (1 mm3 3 or less)or less)
• Tissue must be freshTissue must be fresh
• Double fixation (Double fixation (glutaraldehyde for proteins plus osmium glutaraldehyde for proteins plus osmium tetroxide for lipids [osmium also stains])tetroxide for lipids [osmium also stains])
• Quick dehydration and clearingQuick dehydration and clearing
• Embedding in plastic resins Embedding in plastic resins (Epon or Araldite)(Epon or Araldite)
• Cut with ultramicrotome (Cut with ultramicrotome (glass or diamond knifesglass or diamond knifes) produces 30 ) produces 30 – 50 nm thick specimens– 50 nm thick specimens
• Mounting in copper gridsMounting in copper grids
• Stain with heavy metals
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Tissue Fixation in Electron MicroscopyTissue Fixation in Electron Microscopy
Same principles as in Light Microscopy with the following differences:
Double fixation in glutaraldehyde and osmium tetroxide
Ultra-thin sections (< 90nm thick) from plastic embedded blocks.
Thus, all the original EM pictures are black-white images.
TEST YOURSELF
The agents in which procedure are intended to stabilize tissue structure by coagulating proteins and promoting cross-linking in?
(a) Clearing(b) Dehydration(c) Embedding(d) Fixation
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StainsStains
Nuclei are basophilic, since they are mainly made of DNA
Nuclei are basophilic, since they are mainly made of DNA
Cytoplasm is acidophil, since most proteins are basic in nature
Cytoplasm is acidophil, since most proteins are basic in nature
• Acid dyes stain basic structures: (proteins, membranes, cytoplasm). The commonest acid dye is eosin; it is pink
• Basic dyes stain acid structures: Nucleic acids (RNA, DNA, rER, nuclei). The commonest basic dye is hematoxylin; it is blue
• NOTE: tissues that stain with basic dyes are termed basophilic; tissues that stain with acid dyes are termed acidophilic
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Regular Stains:•Hematoxylin & Eosin (H&E)
•Trichromes (differentiate intercellular structures)
•Iron hematoxylin (for iron-containing cells, like muscle & red blood cells)
Specific stains:Proteoglycans (PAS, cresyl violet, toluidine blue, methylene blue)
Neurons (Nissl, Ag, Osmium, cresyl violet)
Elastic fibers (orcein or resorcein)
Reticular fibers (silver stain)
Blood cells (Romanovsky stains)
Polysaccharides (PAS)
Useful stains in histologyUseful stains in histology
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Haematoxylin-eosin stainingHaematoxylin-eosin staining
• H&E is the most common dye used in the study of histology
• Hematoxylin stains acidic structures (nucleic acids, nuclei, rER) blue
• Eosin stains basic structures (proteins, membranes) pink
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Structures that are not readily stained by hematoxylin or eosin, are called neutrophilic structures.
Which of the following statements is false about the rough endoplasmic reticulum(RER)?
(A) Prominent in cells specialized for protein secretion
(B) Presence of polyribosomes confers basophilia
(C) Presence of polyribosomes confers acidophilia in the cell (D) This organelle can be viewed with a light microscope
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Trichrome stain
• Uses three dyes to differentiate intercellular structures
• Particularly helpful in highlighting red blood cells within blood vessels
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• Gomori trichrome
• Mallory’s trichrome
• Masson’s trichrome
• Red keratin and muscle fibers, • Blue or green collagen and bone, • Light red or pink cytoplasm, and • Dark brown to black cell nuclei
Trichrome stain
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Trichrome stain
Note the blue nuclei, the pink/purple cytoplasm, the red blood cells and the tuquoise material that is non-cellular.
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Trichrome stain
Note:
• Blue nuclei• Pink color in
cytoplasm• Deep red of blood
cells
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Diff btw H&E and Trichrome stain
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Iron-haematoxylin
• Highlights tissue and cells that contain iron, such as muscle & red blood cells
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Specific stains for proteoglycans
• Periodic acid-Schiff (PAS) reaction ALSO USEFUL FOR THE IDENTIFICATION OF POLYSACCHARIDES (Glycogen)
• Cresyl violet
• Toluidine blue
• Methylene blue
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Periodic Acid Schiff (PAS) stainingPeriodic Acid Schiff (PAS) stainingPAS reaction is a method to demonstrate glycogen-like substances.
Periodic acid oxidizes the glycol groups in the glucose residues into aldehyde groups, which then react with Schiff’s reagent, producing an insoluble compound with a reddish purple color.
PAS reaction in a hepatocyte PAS reaction in mucosa of intestine
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Specific stains used for NEURONS
• Nissl (a specialized stain for rER of neurons)
• Silver & Gold (for fibers & cytoskeletal elements)
• Osmic acid (for myelin, a lipid)
• Cresyl violet (proteoglycans)
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Nissl staining
A specialized stain for rough endoplasmic reticulum in neurons
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Silver StainingSilver Staining
Reticular fibers cannot be well seen with H&E staining
Silver ions are deposited on the structures and reduced to silver particles, showing brown to black color, (argyrophilic).
Reticular fibres
Neurofilaments stained with silver salts
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Reticular stain
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Cresyl violetCresyl violet
• Highlights proteoglycans• Most commonly used for staining nervous tissue• This is a violet/purple stain
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ElastinElastin
Elastin stains elastic fibers black
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Verhoff stain for elastin
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Romanovsky Stain for Blood CellsRomanovsky Stain for Blood Cells
Romanovsky stains (Giemsa, Wrights, etc.) are useful for highlighting various types of granules present in developing and mature white blood cells.
Note the following:
• Reddish-brown cytoplasm of red blood cells
• Large (white) cell with deep blue granules
• White blood cells are most readily identified with the use of Romanovsky – type stains
• E g GIEMSA
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The granules in cytoplasm of mast cells are shown in purple color when stained by toluidine blue, a blue dye.
MetachromasiaMetachromasiaPhenomenon where a certain dye shows structures in a different color from that of the dye.
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Toludine blue
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Osmic acid stainingOsmic acid staining
****The effect of osmium tetraoxide is to preserve and stain lipids and proteins.*****
Oil Red O
• Oil Red O is used to stain lipids a red-orange color in unfixed frozen sections.
. Q1. which technique is most commonly used to locate glycogen in cells?(a) Methylene blue staining(b) Periodic acid-schiff(PAS) reaction(c) Enzyme histochemistry
Q 2. frozen sectioning may be required to avoid the removal of which target substance when preparing tissues for paraffin sectioning?(a)Basic proteins(b)Lipids(c)Enzymes(d)carbohydrates
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ARTIFACTS
• Pre-histology• These are features and structures that have
being introduced prior to the collection of the tissues.
• A common example of these include: ink from tattoos and freckles (melanin) in skin samples.
• Post-histology• Artifacts can result from tissue processing.
Processing commonly lead to changes like shrinkage, color changes in different tissues types and alterations of the structures in the tissue.
• Because these are caused in a laboratory the majority of post histology artifacts can be avoided or removed after being discovered.
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Artifacts (not natural occurences)Artifacts (not natural occurences)
The main cause of artifacts is poor fixation
The main cause of artifacts is poor fixation
Some tissues usually present artifacts, & wrong knowledge has been derived from them
Some tissues usually present artifacts, & wrong knowledge has been derived from them
Caused by a bad histological technique
Autolysis
Poor sampling
Shrinkage
Folds
Stain precipitation and dust
Defects in the knife
Caused by a bad histological technique
Autolysis
Poor sampling
Shrinkage
Folds
Stain precipitation and dust
Defects in the knife
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Suprarenal gland showing with autolysis
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Shrinkage and rupture
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Shrinkage
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Folds
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Breaking and notches
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Knife marks
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Edge damage
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Poor fixation
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Dust