Staining and Immunohistochemistry PDF

# STAINING - **STAINING:** Process of applying dyes on the sections to see and study the architectural pattern of the tissue and physical characteristics of the cell. ## METHODS OF STAINING - **Direct Staining:** Process of giving color to the sections using aqueous dye solutions. - **Indirect St...

# STAINING - **STAINING:** Process of applying dyes on the sections to see and study the architectural pattern of the tissue and physical characteristics of the cell. ## METHODS OF STAINING - **Direct Staining:** Process of giving color to the sections using aqueous dye solutions. - **Indirect Staining:** Action of dye is intensified by adding another agent or a mordant. - **Mordant:** Serves as a bridge between tissue and the dye to make staining reaction possible. - **Accentuator:** Accelerates the speed of staining reaction by increasing the staining power and selectivity. - **Blueing:** Alum hematoxylin stains nuclei with red color, which is converted to the familiar blue black when the section is washed in a weak alkali solution. Tap water is usually alkaline enough to produce this color change, but occasionally alkaline solutions such as saturated lithium carbonate, 0.5% ammonia in distilled water, or Scott's tap water substitute are necessary. - **Metallic impregnation:** Process where a specific tissue element is demonstrated by colorless solution of metallic salts which are reduced by the tissues producing an opaque, usually black deposit on the surface of the tissue. - **Negative staining:** Stains are not taken up by their tissue targets. - **Vital staining:** Selective staining of living cell constituents. - **Counterstaining:** Application with a different color for contrast and background. ## STAINS AND STAINING SOLUTIONS ### Natural dyes 1. **Hematoxylin** - Most valuable staining reagent. - Extracted from the core wood of a Mexican tree called _Haematoxylon campechianum_. 2. **Cochineal dyes** - Old histologic dye derived from an extract from the female cochineal bug _Dactylopius coccus costal Coccus cacti_. 3. **Orcein** - Vegetable dye extracted from lichens. 4. **Saffron** - Derived from the dried stigmata of _Crocus sativus_. ### Synthetic dye - **Chromophore:** Substances that are capable of producing visible color. - **Chromogens:** Simple benzene compounds that contain chromophores. - **Auxochrome:** Substances added to chromogen that alters its property. - **Dye:** It should consist of an auxochrome and a chromophore group attached to a hydrocarbon benzene ring. ## COMMON STAINING SOLUTIONS ### 1. HEMATOXYLIN - **Aluminum (Alum) hematoxylin:** - Ehrlich's hematoxylin - Harris hematoxylin - Delafield's hematoxylin - Cole's hematoxylin - Mayer's hematoxylin - Gill hematoxylin - **Iron hematoxylin:** - Weigert's hematoxylin - Heidenhain's hematoxylin - Loyez hematoxylin - Verhoeff's hematoxylin - **Tungsten Hematoxylin:** - Phosphotungstic acid hematoxylin - **Copper hematoxylin** ### 2. EOSIN - Most widely used cytoplasmic stain. Best staining with eosin occurs at pH 4.6 to 5. ### 3. ACID FUCHSIN-PICRIC ACID - Simplest method of differential staining of collagen. ### 4. ACRIDINE ORANGE - Most commonly used fluorochrome to demonstrate DNA and RNA. ### 5. ALCIAN BLUE - Stains acid mucopolysaccharides by forming salt linkages with them. ### 6. ANILINE BLUE - Cytoplasmic stain used for counterstaining of epithelial sections. ### 7. BASIC FUCHSIN - Stains acid-fast organism, mitochondria, differentiation of smooth muscles with the use of picric acid. ### 8. BENZIDINE - Stains hemoglobin ### 9. BISMARCK BROWN - Stains Diphtheria organisms ### 10. MALACHITE GREEN - Use as both decolorizer and a counterstain. ### 11. ORCEIN - Stain for elastic fibers. ### 12. CELESTINE BLUE - Recommended for routine staining of fixed sections. ### 13. CONGO RED - Stain for axis cylinders in embryos. ### 14. CRYSTAL VIOLET - Used to stain amyloid in frozen section. ### 15. IODINE - Stains amyloid, cellulose, starch, carotenes and glycogen. ### 16. JANUS GREEN B - Demonstration of mitochondria. ## ROUTINE H&E STAINING FOR PARAFFIN EMBEDDED SECTIONS **PROCEDURE:** 1. Clear paraffin-embedded section in first xylene bath for 3 mins. 2. Transfer to second xylene bath for 2-3 mins. 3. Immerse in first bath of absolute ethanol for 2 mins. 4. Transfer to a bath of 95% ethanol for 1-2 mins. 5. Rinse with running water for 1 min. 6. Stain with Harris hematoxylin for 5 mins. 7. Wash with running water. 8. Decolorize using acid alcohol for 10-30 secs. 9. Rinse in tap water. 10. Blueing step (ammonia water for 5 mins. or 1% Aq. lithium carbonate until sections appear blue). 11. Wash with running water for 5 mins. 12. Counterstain with 5% Aq. Eosin for 5 mins. (Alcoholic eosin, counterstain for 30 secs, or 1 min.) 13. Dehydrate, clear, and mount. # IMMUNOHISTOCHEMISTRY - Use of antibodies as histological tools for identifying patterns of antigen distribution within an organism or tissue. - Immunohistochemical techniques are now routinely used for the identification of specific or highly selective antigens/epitopes in frozen or paraffin-embedded tissues. ## POLYCLONAL ANTIBODIES: - Produced by immunizing an animal with an immunogen that contains the antigen of interest. ## MONOCLONAL ANTIBODIES: - Products of an individual clone of plasma cells. ## IMMUNOHISTOCHEMISTRY TECHNIQUES - **Direct technique:** Conjugate the primarily antibody directly to the label such as fluorochrome or horseradish peroxidase. - **Indirect technique:** Two or three-step procedure that involves application of unconjugated primary antibody, followed by a labeled antibody directed against the first antibody - **Peroxidase-Antiperoxidase technique:** An indirect antibody enzyme-complex technique where the soluble peroxidase-antiperoxidase complex is bound to unconjugated primary antibody by a second layer of "bridging" antibody, usually a swine anti-rabbit antibody, that then binds to both the primary antibody and the rabbit PAP complex. - **Avidin-biotin complex technique:** Basic sequence of staining consists of primary antibody, biotinylated secondary antibody followed either by preformed avidin-biotin-enzyme complex of the avidin-biotin complex technique or by labeled streptavidin. - **Labeled streptavidin avidin biotin technique:** The staining sequence consists of primary rabbit antibody, biotinylated anti-rabbit immunoglobulin and streptavidin-enzyme conjugate. The color reaction is then developed with appropriate substrate or chromogen, such as horseradish peroxidase. # I. CARBOHYDRATES | Technique | Result | |-----------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------| | PERIODIC ACID SCHIFF, BEST CARMINE | PAS+=magenta red, Nuclei=blue<br>Glycogen=bright red granules,<br>Nuclei=grayish blue/blue<br>Mucin, fibrin=weak red | | LANGHAN'S IODINE METHOD FOR GLYCOGEN | Glycogen= mahogany brown, Tissue constituents=yellow | | ALCIAN BLUE | Acid mucin=blue, Nuclei= red | | METACHROMATIC TOLUIDINE BLUE | Glycosaminoglycans= red purple, Tissue background=blue | | FLUORESCENT ACRIDINE ORANGE | Acid mucopolysaccharide=black, Fungi=greenish red fluorescence, Background=reddish orange fluorescent | | HALE'S DIALYZED IRONE | Acid mucin= dark blue, nuclei= blue | # II. FATS/LIPIDS | Technique | Result | |--------------------|--------| | SUDAN IV | Lipids | | OIL RED O | Dextrin | | OSMIC ACID STAIN | Fats | # III. NUCLEIC ACID | Technique | Result | |-------------------------------|-------------------------------------------------------------------| | FEULGEN TECHNIQUE | DNA=red purple, Cytoplasm=green | | METHYL GREEN | DNA=green/blue green, RNA= rose red | | ACRIDINE ORANGE- FLUORESCENT | DNA= yellow green fluorescent <br> RNA= brick to orange red | # IV. PROTEINS | Technique | Result | |--------------------|----------------------------------------------------------------------| | ALKALINE FAST | Method for basic proteins | | PERACETIC ACID | Cystine, cysteine=blue green | | SAKAGUCHI'S TEST | Arginine= orange red | # V. ENZYMES | Technique | Result | |------------------------|-------------------------------------------------------------------------------| | GOMORI CALCIUM | ALP= brownish black, Nuclei=green | | GOMORI LEAD | ACP=black, Nuclei=green | | LEAD METHOD | 5' nucleotidase= blackish brown deposits | | A-NAPHTHYL ACETATE | Esterase= reddish brown, Nuclei=green | | INDOXYL ACETATE | Esterase activity=blue, Nuclei=red | | TETRAZOLIUM | Monoamine oxidase=bluish black | # VI. CONNECTIVE TISSUE | Technique | Result | |-------------------------------|------------------------------------------------------------------------------------------------------| | GOMORI'S SILVER IMPREGNATION | Reticulin fiber= black | | VAN GIESON'S | Collagen= pink/deep red, Nuclei=brownish black | | MASSON'S TRICHROME STAIN | Muscle, RBC, keratin=red, Nuclei= blue/black | | WEIGERT'S | Elastic fibers= dark blue or blue black on clear background | | VERHOEFF'S | Elastic fibers= black, Nuclei= gray to black | | ORCEIN | Elastic fiber=dark brown, Nuclei= blue | | KAJIAN'S TECHNIQUE | Elastic fiber=bright red, Fibrin, CT=Dark blue, RBC=orange yellow | | PTAH STAIN | Fibrin, neuroglia, muscle striation, amoeba=dark blue | | METHYL VIOLET-CRYSTAL VIOLET | Amyloid=purplish red, Nuclei, cytoplasm, CT= shades of violet | | KRAJIAN AMYLOID STAIN | Amyloid= red on clear background, Nuclei=blue | # VII. CENTRAL NERVOUS SYSTEM | Technique | Result | |---------------------------------|----------------------------------------------------------------------------------------------| | BIELSCHOWSKY'S TECHNIQUE | Neurofibril, axon, dendrites=black/grayish bg. | | SEVIER-MUNGER | Neural tissues | | CRESYL-FAST VIOLET | Paraffin sections | | LUXOL FAST BLUE-H&E STAIN | Myelin=blue green | | LUXOL FAST BLUE-PAS STAIN | Myelin=blue/green | | WEIL'S METHOD | Myelin sheath-black, background=yellow | | CAJAL'S GOLD SUBLIMATE | Astrocytes=black on a light brownish bg. | | WEIGERT-PAL | Normal myelin sheath=blue black, cells=brown | | KLUVER AND BARRERS LUXOL FAST BLUE | Myelin with Nissi counterstain | # VIII. TISSUE PIGMENTS AND DEPOSITS | Technique | Result | |-------------------------------------------|---------------------------------------------------------------------------------| | PERL'S PRUSSIAN BLUE | Hemosiderin and ferric salts | | SCHMORI'S FERRIC-FERRICYANIDE | Reducing substances | | MALLORY'S FUCHSIN STAIN | Hemofuscin pigment | | MASSON-FONTANA | Melanin and argentaffin cell's granule | | VON KOSSA'S SILVER NITRATE | Calcium | | LINQUIST'S MOD. RHODANINE | Copper | | CALCIUM DYE LAKE | Calcium salts= intense reddish orange | | MOD. FOUCHET'S TECHNIQUE | Liver bile pigments=emerald to blue green | | GOMORI'S ALDEHYDE FUCHSIN | Lipofuscin=purple | # IX. MICROORGANISMS | Technique | Result | |---------------------------|--------------------------------------------------------------------| | WADE-FITE | Leprosy bacilli and nocardia | | TOLUIDINE BLUE | Helicobacter=dark blue | | DIETERLE | Legionella pneumophila | | LEVADITI'S MTD. | Spirochetes | | WARTHIN-STARRY | Spirochetes | | MOD. STEINER AND STEINER | Spirochetes | | GROCOTT METHENAMINE SILVER | Fungi=black | | RAPID GIEMSA STAIN | Bacteria=blue | | ZIEHL-NEELSEN MTD. | AFB=red, Cells, nuclei=blue, RBC=pink |