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Connective & Muscle
Staining
| Question | Answer |
|---|---|
| What are the 3 components of connective tissue? | Fibers, cell, & amorphous ground substance |
| What are the 4 connective tissue types? | Connective tissue proper (elastic, collagen, reticulin) cartilage, bone, blood |
| Ground substance of connective tissue is a_ | Mucopolysaccharide |
| Collagen fibers: | Strength, eosinophilic, birefringent, show cross-striation under EM, Masson, Gomori, van Gieson |
| Elastic fibers: | Stretch, not in H&E, Verhoeff, Gomori |
| Reticular fibers: | Form delicate networks, demo w/ argyrophilic rxns, Gomori, Gordon & Sweets |
| Cell found in connective tissue proper include: | blood, adipose, plasma, mast, mesenchymal, macrophages, fibroblasts |
| Masson Trichrome stain purpose | Differentiate collagen & smooth muscle tumors, ID collagen increase due to disease (e.g. cirrhosis of liver) |
| Masson Trichrome stain facts | Bouin; 10% NBF ok QC: most tissue has internal control, can use fallopian tube, uterus, small intestine, appendix |
| Masson Trichrome stain solutions | Biebrich Scarlet-Acid fuchsin + Bouin + Weigert + aniline blue + acetic acid + phosphomolybdic/phosphotungstic acid |
| Masson Trichrome stain conventional results | nuclei: black - collagen, mucus: blue - cytoplasm, keratin, muscle fibers: red |
| Biebrich Scarlet-Acid Fuchsin solution | Biebrich scarlet + acid fuchsin + glacial acetic acid |
| Phosphomolybdic/ phosphotungstic acid solution | phosphomolybdic acid + phosphotungstic acid + dH2O |
| Aniline blue solution | aniline blue + glacial acetic acid + dH2O |
| Masson Trichrome stain microwave results | nuclei: dark blue - cytoplasm, keratin, epithelium, muscle fibers: red - collagen & mucus: blue |
| Masson Trichrome can also be counterstained w/_ | Light green, best counterstain if collagen predominant, aniline blue better if little collagen |
| Gomori One-Step Trichrome stain facts | To ID incr. in collagenous fibers, differentiate between collagen & smooth muscle, any fixative, Bouin is mordant QC: most tissue has internal control, can use uterus, fallopian tube small intestine, appendix |
| Gomori One-Step Trichrome stain solutions | Gomori Trichrome + Bouin + Weigert + acetic acid |
| Gomori One-Step Trichrome stain results | nuclei: black - cytoplasm, keratin, muscle fibers: red - collagen & mucus: green or blue |
| Gomori Trichrome stain solution | chromotrope 2R (plasma stain) + fast-green FCF, light green, or aniline blue (connective stain) + glacial acetic acid + phosphotungstic acid (muscle & cytoplasm red) + dH2O |
| van Gieson Picric Acid-Acid Fuchsin stain facts | Used as counterstain, esp. for Verhoeff, demo collagen, any fixative, QC: most tissue has internal control, can use uterus, fallopian tube small intestine, appendix |
| van Gieson Picric Acid-Acid Fuchsin stain solutions | van Gieson - acid fuchsin (stains collagen) + picric acid, sat'd (stains muscle) + (can add HCl to sharpen differentiation) Weigert |
| van Gieson Picric Acid-Acid Fuchsin stain results | nuclei: black - collagen: brilliant red - muscle & cytoplasm: yellow |
| What type of hematoxylin is used in trichrome procedures? | Iron hematoxylin, b/c more resistant to decolorization by acidic dyes than aluminum hematoxylins |
| Verhoeff Elastic stain facts | To demo pathologic changes in elastic fibers, 10% NBF, Zenker QC: aorta, artery |
| Verhoeff Elastic stain solutions | Verhoeff (dye lake) + van Gieson + ferric chloride (excess mordant, regressive) + sodium thiosulfate |
| Verhoeff Elastic stain results | elastic fibers: blue-black to black - nuclei: blue to black - collagen: red - other: yellow |
| Pathologic changes in elastic fibers include: | Thinning, loss b/c arteriosclerotic changes, reduplication, breaks, splitting b/c other vascular diseases |
| Lugol iodine solution | iodine + potassium iodide + dH2O |
| Verhoeff elastic stain solution | alcoholic hematoxylin + ferric chloride (mordant & ox) + lugol iodine (mordant & ox) |
| Aldehyde Fuchsin elastic stain (Gomori) facts | To demo pathologic changes in elastic fibers, 10% NBF; avoid chromate fixatives, formalin, Bouin-fixed, colorless background, mercury-fixed, lilac background, QC: aorta, artery, skin, can stain β cells (pancreatic islets, pit) |
| Aldehyde Fuchsin elastic stain (Gomori) solutions | Aldehyde fuchsin soln. + light green working |
| Aldehyde Fuchsin elastic stain (Gomori) results | elastic fibers: deep blue to purple - other: green |
| Aldehyde Fuchsin solution | pararosaniline (basic fuschin) + paraldehyde + 70% + HCl Stable 3-4 weeks @ 4°C |
| Light green stock solution | light green SF yellowish + glacial acetic acid + dH2O |
| Russell Modification of Movat Pentachrome stain purpose | Demo elastic, collagen, mucin, muscle, fibrin |
| Russell Modification of Movat Pentachrome stain facts | 10% NBF, acetic formalin sublimate QC: lung, skin, colon |
| Russell Modification of Movat Pentachrome stain solutions | Alcian blue (acid mucosubstances) + alkaline alcohol + alcoholic Safran (collagen) + hematoxylin soln. (elastic) + ferric chloride (diff) + sodium thiosulfate + phosphotungstic acid + Crocein Scarlet-acid fuchsin |
| Russell Modification of Movat Pentachrome stain results | nuclei, elastic fibers: black - ground substance, mucin: blue - Cryptococcus neoformans: bright blue - fibrinoid, fibrin: intense red - muscle: red - collagen: yellow |
| Acetic formalin sublimate solution | formaldehyde + glacial acetic acid + dH2O |
| Alcoholic Safran solution | Safran du Gatinais + absolute alcohol |
| Crocein Scarlet-acid fuchsin solution | Crocein scarlet + acetic acid + H2O acid fuchsin + acetic acid + H2O |
| Hematoxylin solution | abs. alcoholic hematoxylin + abs. alcohol + ferric chloride + iodine-iodide |
| Absolute alcoholic hematoxylin 10% | hematoxylin + absolute alcohol |
| Alkaline alcohol | NH4OH + 95% |
| Toning: | When bound metallic silver is treated w/ gold chloride & color changes from brown to black as metallic silver is replaced by metallic gold |
| Unreduced silver is removed with? | Sodium thiosulfate (hypo) |
| Gomori stain & Gordon & Sweets for reticular fibers facts | To demo reticular fibers for diagnosis tumors, liver disease, 10% NBF QC: liver |
| Gomori stain for reticular fibers solutions | ferric ammonium sulfate (sensitizer) + formalin (red) + ammoniacal silver soln. + gold chloride + potassium permanganate (ox.) + potassium metabisulfite (remove K. per.) + nuclear-fast red |
| Gomori stain for reticular fibers results | reticulin: black - collagen: taupe |
| Ammoniacal silver solution | silver nitrate + KOH |
| Gordon & Sweets stain for reticular fibers solutions | ferric ammonium sulfate (sensitize) + formalin (red.) + ammoniacal silver + gold chloride + sodium thiosulfate + potassium permanganate (ox.) + oxalic acid 1% (remove K. per.) + nuclear-fast red |
| Gordon & Sweets stain for reticular fibers results | reticulin: black |
| Mallory PTAH technique for cross-striations & fibrin purpose | Demo cross-striations & fibrin to diagnose rhabdomysarcomas, or nemaline rods from disease |
| Mallory PTAH technique for cross-striations & fibrin facts | Zenker; 10% NBF ok paraffin 4-6 μm QC: skeletal or cardiac for cross-striations, section w/ fibrin for fibrin, cerebral cortex for glial fibers polychrome stain |
| Mallory PTAH technique for cross-striations & fibrin solutions | PTAH soln. + Gram iodine + sodium thiosulfate + potassium permanganate + oxalic acid |
| Mallory PTAH technique for cross-striations & fibrin results | cross-striations, fibrin, nuclei: blue - collagen: red-brown |
| PTAH solution | hematoxylin + phosphotungstic acid + H2O |
| Periodic acid-methenamine silver microwave procedure for basement membranes purpose | Best delineates glomerular basement membrane |
| Periodic acid-methenamine silver microwave procedure for basement membranes facts | 10% NBF; avoid mercury fixatives paraffin 2 μm QC: kidney has internal control |
| Periodic acid-methenamine silver microwave procedure for basement membranes solutions | periodic acid 1% (ox. carbs to aldehydes) + methenamine silver working + gold chloride + sodium thiosulfate + light green working |
| Periodic acid-methenamine silver microwave procedure for basement membranes results | basement membrane: black + background: green |
| Methenamine silver working solution | methenamine (make soln. alkaline) + silver nitrate + Borax (buffer) + H2O |
| Borax solution | sodium borate + H2O |
| Oil Red O method for neutral fats purpose | Demo neutral lipids in frozens, demo fat emboli, or injury to fatty body area |
| Oil Red O method for neutral fats solutions | oil red O + Harris w/ acetic acid + ammonia water |
| Oil Red O method for neutral fats results | fat: intense red - nuclei: blue |
| Oil Red O working solution | oil red O + isopropanol + H2O |
| Oil red O must be coverslipped with? | Aqueous mounting medium, seal w/ nail polish |
| Sudan Black B in propylene glycol purpose | Demo neutral lipids, differentiate granulocyte precursors from leukocytes turning to lymphocytes or monocytes |
| Sudan Black B in propylene glycol & Oil red O method for neutral fats facts | 10% NBF, (or post-fix w/) calcium-formalin; avoid alcoholic fixatives frozen 10 μm QC: most tissue has fat |
| Sudan Black B in propylene glycol solutions | propylene glycol + Sudan Black + nuclear-fast red |
| Sudan Black B in propylene glycol results | fat: blue-black - nuclei: red |
| Sudan Black B staining solution | Sudan black B + propylene glycol |
| OsO4 paraffin procedure for fat purpose | Demo fat in paraffin |
| OsO4 paraffin procedure for fat facts | 10% NBF Technique: section =< 2mm, stain, process QC: Most tissue has fat |
| OsO4 paraffin procedure for fat solution | OsO4 + periodic acid |
| OsO4 paraffin procedure for fat results | fat: black - other: depends on stain |
| What is used to stain lipids treated by OsO4 paraffin procedure for fat? | Routine H&E or any special stain, Masson trichrome good counterstain |
| Toluidine blue for mast cells facts | To demo mast cells, 10% NBF QC: section w/ mast cells |
| Toluidine blue for mast cells solution | Toluidine blue + H2O |
| Toluidine blue for mast cells results | mast cells: deep violet - background: blue |
| PTAH stains tissue components: | Red-brown/salmon, components will lose color if prolonged water or alcohol washing so dehydration after staining must be rapid |
| Blood cells may be demonstrated with? | Giemsa |
| Snook reticulin stain solutions | potassium permanganate (ox.) + uranyl nitrate (sensitize) + ammoniacal silver + formaldehyde (red.) + gold chloride + sodium thiosulfate |
| Laidlaw reticulin stain solutions | potassium permanganate (ox.) + lithium silver (impregnate) + formaldehyde (red.) + gold chloride + sodium thiosulfate |
| Nasher & Shanklin reticulin stain solutions | potassium permanganate w/ sulfuric acid (ox.) + AgNo3 (sensitize) + ammoniacal silver + formaldehyde w/ abs. alcohol (red.) + gold chloride + sodium thiosulfate |
| Wilder reticulin stain solutions | phosphomolybdic acid (ox.) + uranyl nitrate (sensitize) + ammoniacal silver + formaldehyde w/ uranyl nitrate (red.) + gold chloride + sodium thiosfulate |
| Silver techniques steps: | 1) ox. (glycol groups of sugars to aldehydes) 2) sensitization 3) silver impregnation 4) reduction (residual silver diamine to metallic silver) 5) toning 6) unred. silver removed 7) maybe counterstain |
| Granular deposition of silver in reticulin staining is likely due to? | Old reagents, especially sensitizers |
| Orcein elastic stain | Orcein in acidified alcohol, elastic fibers - brown, less intense than Verhoeff |
| Resorcin fuchsin elastic stain | Resorcin fuchsin in acidified alcohol, counterstain - van Gieson, looks similar to Verhoeff |
| Miller technique for elastic | Victoria blue 4R + new fuchsin + crystal violet + H2O + resorcin + dextrin + ferric chloride + HCl |
| Trichrome staining basics | 2+ contrasting dyes used to selectively stain, usu. to differentiate collagen from muscle, @ acid pH, usu. dilute acetic acid to accentuate attachment of dye to proteins, plasma dye stains cytoplasm, fiber dye stains collagen, Bouin best b/c mordants |
| Trichrome staining colors | Plasma stain red, fiber dye blue or green, nuclear counterstain provides 3rd color, aluminum hematoxylins exhibit atypical colors @ acid pH so must remove or use iron hematoxylin |
| Masson vs. Gomori trichromes | Masson multi-step - acid dye, then aid to destain collagen, stain collagen, stain nuclei, Gomori one-step - cytoplasmic & fiber stain in one solution, if either doesn't show clear differentiation & distinct color may be old reagents |
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