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Basic Histology

basic questions on fixation, H&E, special stains, and other histology topics

QuestionAnswer
Ehrlich Hematoxylin: progressive/regressive, mordant, ripening, uses/special considerations Usu. regressive, but can be used progressively, potassium aluminum sulfate, ripened naturally or chemically, gives crisp nuclear staining.
Delafield Hematoxylin: progressive/regressive, mordant, ripening, uses/special considerations regressive, ammonium aluminum sulfate, ripens naturally, alcohol preserves, glycerol prevents overoxidation and rapid evaporation
Mayer Hematoxylin: progressive/regressive, mordant, ripening, uses/special considerations Progressive, potassium aluminum sulfate, sodium iodate ripens, citric acid adjusts pH, chloral hydrate prevents precipitates. Good for AEC immuno techs b/c it does not contain alcohol, which would dissolve reaction product. Slow, but crisp nuclear stainin
Gill Hematoxylin: progressive/regressive, mordant, ripening, uses/special considerations Progressive, aluminum sulfate, sodium iodate, ethylene glycol prevents surface precipitates, can be used immediately, but better left to ripen for a week at 37C., stains goblet cells, esp. in mucin. GillIII is used on Glycol Methacrylate sections
Weigert Hematoxylin progressive/regressive, mordant, ripening, uses/special considerations Progressive, Ferric Chloride (Fe) is mordant and oxidizer, not for routine stains, resists acid decolorization
What are some nuclear stains besides hematoxylin? safranin, nuclear fast red, toluidine blue o, thionin, methylene blue
Harris Hematoxylin: progressive/regressive, mordant, ripening, uses/special considerations progressive or regressive, ammonium aluminum sulfate, ripen with mercuric oxide or sodium iodate, filter just before use,add acetic acid to use progressively, can be over differentiated easily
Acetone: Action, advantages/disadvantages, uses, special considerations non-additive/coagulant, overhardens and shrinks tissue, can be used to demonstrate enzymes, esp. good for rabies
Acetic acid: Action, advantages/disadvantages, uses, special considerations non-additive/coagulant, does not fix or destroy carbohydrates, does not fix lipids, causes swelling, precipitates and preserves nucleoproteins (DNA/RNA), caustic, corrosive
Alcohols: Action, advantages/disadvantages, uses, special considerations non-additive, coagulant, overhardens and shrinks tissue, good for blood smears, and to preserve water soluble tissue elements such as glycogen and urates. dissolves lipids
Glutaraldehyde: Action, advantages/disadvantages, uses, special considerations additive/noncoagulant, fixes at the rate it penetrates, but penetrates slowly and poorly, preserves ultrastructure well, so is good for EM, breaks down with exposure to oxygen, incompatible with oxidizers and alkali
Osmium tetroxide: Action, advantages/disadvantages, uses, special considerations additive/noncoagulant, binds to fat, can only penetrate a few cell layers, preserves ultrastructure so is good for EM, usu. as a postfixative, vaporizes readily and can fix corneas, eye and nasal mucosa
potassium dicromate: Action, advantages/disadvantages, uses, special considerations additive/noncoagulant, renders lipids insoluble, preserves mitichondria but dissolves DNA, leaves tissue soft, but also able to shrink in subsequent processing steps, highly toxic, carcinogenic, and corrosive
mercuric chloride: Action, advantages/disadvantages, uses, special considerations additive/coagulant, extremely toxic/corrosive, so no longer used alone, penetrates poorly, does not distort cells, but leave tissue able to shrink during processing, can often be substituted with zinc salts
paraformaldehyde: Action, advantages/disadvantages, uses, special considerations additive/noncoagulant, polymerized form of formaldehyde, used in EM, when very pure formaldehyde is needed, heat do dissociate to formaldehyde
B-5: composition, advantages/disadvantages, uses, special considerations mercuric chloride, formaldehyde, sodium acetate. hematopoetic, good for lymphoreticular tissue, good with many special stains and antigens
Bouins: composition, advantages/disadvantages, uses, special considerations picric acid, formaldehyde, acetic acid. swelling of acetic acid is balanced by shrinking of picric acid, good for trichrome staining, preserving tissue with soft structures, GI specimens, endocrine tissue. lyses erythrocytes, dissolves calcium and iron
Carnoy's: composition, advantages/disadvantages, uses, special considerations chloroform, acetic acid, absolute alcohol. lyses erythrocytes, dissolves lipids, rapid, preserves glycogen, good nuclear detail, but causes excessive shrinkage and hardening. good for cytology
Helly solution: composition, advantages/disadvantages, uses, special considerations mercuric chloride, potassium dicromate, sodium sulfate, formaldehyde. preserves erythrocytes, good for most stains except silver. extremely toxic due to mercury
Zenker Solution: composition, advantages/disadvantages, uses, special considerations mercuric chloride, patassium dichromate, sodium sulfate, acetic acid. lyses erythrocytes, good nuclear detail, good for most stains except silver, extremely toxic due to mercury
Hollande Solution: composition, advantages/disadvantages, uses, special considerations copper acetate, picric acid, formaldehyde, acetic acid. decalcifies small deposits, good for GI biopsies, lyses erythrocytes less than Bouin's. toxic
Zamboni PAF: composition, advantages/disadvantages, uses, special considerations picric acid, paraformaldehyde, sodium hydroxide, mono- and di- basic sodium phosphate. good all around fixative, allows secondary fixation with osmium tetroxide, good for EM
Orth Solution: composition, advantages/disadvantages, uses, special considerations potassium dichromate, formaldehyde, sodium sulfate. demonstrates chromaffin granules,
What is the Turnbull Blue stain used for, and what control do you use? To detect ferrous iron (Fe3+), sections containing ferrous iron
What are the major reagents used in the Turnbull Blue reaction, and what is the purpose of each? Potassium ferricyanide-hydrochloric acid: reacts with ferrous iron. nuclear fast red: counterstain
What are common errors/problems with the Turnbull reaction? Non specific staining will occur if the reagents or glassware are contaminated with anything metallic, metallic foreceps are used
What is the purpose of the Hall's Bilirubin stain, and what control do you use? to detect the presence of bilirubin, it is reduced to biliverdin. use section containing bilirubin, such as an obstructed bile duct
what are the main reagents and their purpose in Hall's bilirubin stain? Fouchet Reagent: trichloracetic acid & ferric chloride-oxidizes bilirubin to biliverdin
What is the the Grimelius stain (aka Pascual's method) used for, and what control do you use? to detect argyrophil granules in neurosecretory tumors. use argyrophil positive tumor or small intestine
what are the major reagents used in the Grimelius stain (aka Pascual's method), and what is their purpose? acetic-acid acetate buffered silver: bind to argyrophil granules. developer-hydroquinone & sodium acetate: reduces silver to visible form
What are common errors/problems with the Grimelius/Pascual's method? typical silver stain shit. best of luck to you
What is Schmorl's method used for, and what control do you use? to detect the presence of reducing substances in tissue, including melanin, argentaffin granules, and formalin pigment using the Turnbull Blue reaction, use sections containing argentaffin granules or melanin
What are the major reagents in Schmorl's method, and what is their purpose? ferric chloride-potassium ferricyanide: reduces substances and stains them blue with the Turnbull blue reaction. Mayer mucicarmine: counterstain, metanil yellow: counterstain
What is the purpose of the Rhodanine stain? Detect the presence of copper, use a section containing copper
What are the major reagents in the Rhodanine method and their purpose? Rhodanine solution: binds to copper. mayer hematoxylin: nuclear stain.
What are common errors/problems with the Rhodanine stain? Copper color may fade after coverslipping, overstaining with hematoxylin can mask copper
What method is used to demonstrate urates? Gomori methenamine silver
What is a common problem with the GMS for urates? calcium in large deposits may also stain. also, urates are birefringent under a polarizing microscope on an H&E section
What is the Von Kossa stain used for, and what control do you use? detect calcium deposits, use a section containing calcium
what are the major reagents used in the Von Kossa Stain and their purpose? silver nitrate: binds anions in calcium. bright light: reduces silver to visible form. sodium thiosulfate: reduces unreduced silver. nuclear fast red: counterstain
What are common problems/errors with the Von Kossa stain? artificial light, insufficient time in the silver solution, and fixation in unbuffered formalin may reduce staining
What is the Alizarin Red S stain used for, and what control do you use? detect the presence of calcium, use a section containing calcium
what are the major reagent in the alizarin red s stain and their purpose? alizarin red s: stains calcium cations
What is the purpose of the Prussian Blue stain, and what control do you use? To detect excesses of ferric iron (Fe+3)in tissue, use sections containing ferric iron, such as bone marrow
What are the major reagents used in the Prussian blue stain? potassium ferrocyanide-hydrochloric acid: this acidic solution binds to ferric ions to form and insoluble blue compound known as Prussian blue
What are common problems/errors associated with the Prussian blue reaction? Non metallic foreceps must be used, and if glassware or reagents are contaminated by any metallic substance, non specific blue staining will occur throughout the slide.
What is the Churukian Schenk stain for, and what control do you use? to detect argyrophil granules in neurosecretory tumors, use section containing an argyrophil positive carcinoid tumor
What are the major reagents in the Churukian Schenk stain and their purpose? citric-acid sodium citrate silver nitrate-impregnation (pH 3.8). bodian developer: socium sulfite, hydroquinone-developer.
What is the purpose of the Fontana Masson stain, and what control do you use? to detect melanin and argentaffin granules of neurosecretory tumors. use skin for melanin, small intestine for argentaffin granules
what are the major reagents in the Fontana Masson stain and their purpose? fontana ammoniacal silver solution-impregnate. gold chloride-toner. sodium thiosulfate-reduces unreduced silver. nuclear fast red-counterstain
What are common problems/errors with the Fontana Masson stain? use chemically clean glassware and non-metallic foreceps. overstaining will mask the contrast.
What is the purpose of Masson's trichrome stain, and what control do you use? differentiate between collagen and muscle, and identify an increase in collagenous tissue, as in cirrhosis of the liver. control is internal
what are the major reagents in Masson's trichrome stain, and what is their purpose? bouin's-mordant. weigert's iron hematoxylin. beibrich scarlet acid fuchsin-stains muscle, cytoplasm, and collagen. phospho-tungstic/molybdic acid-destains collagen but not muscle. aniline blue-stains collagen and mucin. acetic acid-differentiator
What are common problems associated with Masson's Trichrome stain? use iron hematoxylin, as it is more resistant to acid decolorization than aluminum. pale red staining indicates old reagents. pale blue staining indicates overdifferentiation. sections not fixed/postfixed in bouin's will stain poorly
What is the purpose of the Van Gieson picric acid stain, and what control do you use? differentiate between collagen, and muscle and cytoplasm. internal control
What are the major reagents in the Van Gieson stain and their purpose? Weigert's hematoxylin-nuclear stain. Van Gieson solution-contains acid fuchsin and picric acid, picric acid make the solution acidic enough to only stain muscle and cytoplasm and not collagen.
What are common problems/errors in the Van Gieson stain? if the picric acid used is not saturated, or the pH isn't low enough, differentiation will be insufficient. adding hydrochloric acid will sharpen stain.
What is the purpose of Russel/Movat pentachrome, and what control do you use? Demonstrate mucin, collagen, elastic fibers, muscle, and fibrin. use lung, colon, or skin.
What are the major reagents in the Russel/Movat pentachrome stain and their purpose? alcian blue-mucin&ground substance. alkaline OH-makes AB insoluble. verhoeff sol.-elastin. Fe chloride-differentiate. sodium thio-remove iodine. crocein scarlet acid-muscle&fibrin. phosphotungstic acid-destain coll.&cyto. safran-restain collagen
What are common problems with the Russel/Movat pentachrome? no one uses it
What is the purpose of Gomori's Aldehyde Fuchsin, and what control do you use? to demonstrate elastic fibers, use aorta, cross section of muscular artery, or skin
What are the major reagents used in Gomori's aldehyde fuchsin, and their purpose? aldehyde fuchsin: basic fuchsin, ethyl alcohol, paraformaldehyde, hydrochloric acid-stains elastic fibers. light green-counterstain
What are some commmon problems with Gomori's aldehyde fuchsin? use fresh paraformaldehyde in preparing fuchsin. the fuchsin stain deteriorates fairly quickly. staining times may need to be increased if old aldehyde fuchsin is used
What is the purpose of Gomori's trichrome stain, and what control do you use? to differentiate between collagen and muscle, control is internal
what are the major reagents in Gomori's trichrome, and their purpose? Bouins-mordant, Weigerts-mordant, Gomoris: chromotrope2R, aniline blue, phosphotungstic acid-chromotrope stains connective tissue, phos. acid prevents collagen from taking up the chromotrope, and aniline blue stains the collagen. acetic acid-differentiate
What are common problems/errors with Gomori's trichrome? tissue not fixed/postfixed in Bouins will not stain properly. lowering the pH of the trichrome will intensify staining
What is the purpose of the methyl green pyronin y stain, and what control do you use? differentiate DNA and RNA, identify plasma cells and immunoblasts in cells. use section containing many plasma cells
What are the major reagents used in the methyl green pyronin stain, and their purpose? Methyl green pyronin Y solution-DNA stains with methyl green, RNA stains with pyronin
What are common problems/errors with the methyl green pyronin stain? strong acids and certain fixatives will cause DNA to lose its ability to bind methyl green. Pyronin is not specific for RNA, cartilage, osteoid, keratin, eosinophil granules, and mast cell granules will also stain.
What is Mallory PTAH used for, what is the best fixative, and what control do you use? demonstrate cross striations in muscle or fibrin, use longitudinal section of skeletal or cardiac muscle, or section with fibrin. demonstrate glial cells, use cerebral cortex. fix in Zenker
What are the major reagents in Mallory's PTAH, and their purpose? Phosphotungstic Acid Hematoxylin-there is an excess of PTA. the PTA forms a blue lake with the hematoxylin and is taken up by the cross striations and fibrin, or glial cells. the excess PTA stains other cell components salmon to red-brown
What are common problems/errors with Mallory's PTAH? formalin fixed sections need to be mordanted in zenker, but staining still will not be as good as if originally fixed in zenker. store reagents in dark bottles or they will weaken.
What is Verhoeff's stain used for, and what control do you use? To demonstrate elastic fibers, use a section of aorta, cross section of muscular artery, or skin
What are the major reagents in Verhoeff's stain and their purpose? Verhoeff's stain: alcoholic hematoxylin, Fe chloride, Lugol Iodine (mordant/oxidizer)-stains elastic. ferric chloride-differentiates. sodium thiosulfate-removes excess iodine. Van gieson-counterstain
What are common problems with the Verhoeff stain? it is easy to overdifferentiate, so be careful-too little is better than too much. do not overstain in van gieson, because picric acid will further differentiate. to prepare verhoeff stain, reagents must be added in order, with mixing between each step.
What is the toluidine blue stain for, and what control do you use? Identify mast cells, use a section containing mast cells
What are the major reagents in toluidine blue and their purpose? toluidine blue-metachromatic stain, stains mast cell pink, and background blue.
What is the Periodic acid methenamine silver (PAM) stain for, and what control do you use? demonstrate basement membranes, use section of kidney
What is the difference between the PAM and GMS> PAM uses periodic acid where GMS uses chromic acid because it is weaker and will not over oxidize the carbohydrates in the basal lamina