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Staining

Connective & Muscle Tissue

QuestionAnswer
What are the 3 components of connective tissue? Fibers, cell, & amorphous ground substance
What are the 4 connective tissue types? Connective tissue proper, cartilage, bone, blood
Ground substance of connective tissue is a_ Mucopolysaccharide
What are the 3 fibers of connective tissue proper? Collagen, elastic, & reticular
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
Fibroblasts: Most common cell, produce the fibers, not demo'd
Mesenchymal cells: May be indistinguishable from fibroblasts, undifferentiated, develop as needed, not demo'd
Adipose cells: Synthesize & store lipid, not preserved in paraffin
Mast cells: Secretory granules can mask stains, contain histamine & heparin, metachromatic if stained w/ T blue
Basement membrane: basal lamina, support for epithelium, demo'd by carb (b/c glycoproteins) & silver stains
What are the 3 types of muscle? Skeletal, cardiac, & smooth
Skeletal muscle: Striated voluntary, long multinucleated fibers, PTAH demos cross striations, peroxidase demo w/ monoclonal antibodies
Cardiac muscle: Striated involuntary, may branch, single central nucleus
Smooth muscle: Nonstriated involuntary, long, tapered, single central nucleus
Masson Trichrome stain purpose Differentiate collagen & smooth muscle tumors, ID collagen increase due to disease (e.g. cirrhosis of liver)
Masson Trichrome stain facts Fixative: 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 (v. acidic, stains everything) Bouin Weigert aniline blue (stain collagen) acetic acid 1% phosphomolybdic/phosphotungstic acid (diffuse nonspecific acid dye)
Masson Trichrome stain conventional results nuclei - black collagen, mucus - blue cytoplasm, keratin, muscle fibers - red
Biebrich Scarlet-Acid Fuchsin solution Biebrich scarlet, 1% aq. acid fuchsin, 1% aq. 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, 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 purpose ID increase in collagenous fibers, differentiate between collagen & smooth muscle
Gomori One-Step Trichrome stain facts Fixative: any; 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 0.5%
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 purpose Used as counterstain, esp. for Verhoeff, demo collagen
van Gieson Picric Acid-Acid Fuchsin stain facts Fixative: any QC: most tissue has internal control, can use uterus, fallopian tube small intestine, appendix
van Gieson Picric Acid-Acid Fuchsin stain solutions van Gieson Weigert
van Gieson Picric Acid-Acid Fuchsin stain results nuclei - black collagen - brilliant red muscle & cytoplasm - yellow
van Gieson Picric Acid-Acid fuchsin solution acid fuchsin 1% (stains collagen) picric acid, sat'd (stains muscle, lowers pH) (can add HCl to sharpen differentiation)
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 & Aldehyde Fuchsin elastic stain purpose Demo pathologic changes in elastic fibers
Verhoeff Elastic stain facts Fixative: 10% NBF, Zenker QC: aorta, artery
Verhoeff Elastic stain solutions Verhoeff (dye lake) van Gieson ferric chloride (excess mordant to differentiation - regressive) sodium thiosulfate 5% (remove excess iodine)
Verhoeff Elastic stain results elastic fibers - blue-black to black nuclei - blue to black collagen - red other elements - 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 5% ferric chloride 10% lugol iodine
Aldehyde Fuchsin elastic stain (Gomori) facts Fixative: 10% NBF; avoid chromate fixatives, formalin, Bouin-fixed show colorless background, mercury-fixed show lilac background, QC: aorta, artery, skin
Aldehyde Fuchsin elastic stain (Gomori) solutions Aldehyde fuchsin solution light green working
Aldehyde Fuchsin elastic stain (Gomori) results elastic fibers - deep blue to purple other elements - green
Aldehyde Fuchsin solution pararosaniline (basic fuschin) paraldehyde 70% HCl
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 Fixative: 10% NBF, acetic formalin sublimate QC: lung, skin, colon
Russell Modification of Movat Pentachrome stain solutions Alcian blue 1% (stain acid mucosubstances) alkaline alcohol alcoholic Safran (stain collagen) hematoxylin solution (stain elastic) ferric chloride 2% (differentiation) sodium thiosulfate phosphotungstic acid 5% 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 water acid fuchsin acetic acid water
Hematoxylin solution abs. alcoholic hematoxylin 10% abs. alcohol ferric chloride 10% 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 stain for reticular fibers purpose Demo reticular fibers for diagnosis tumors, liver disease
Gomori stain & Gordon & Sweets for reticular fibers facts Fixative: 10% NBF QC: liver
Gomori stain for reticular fibers solutions ferric ammonium sulfate 2% (sensitizer) formalin (red. silver) ammoniacal silver solution gold chloride 0.2% potassium permanganate 0.5% (ox.) potassium metabisulfite 2% (remove K. per.) nuclear-fast red
Gomori stain for reticular fibers results reticulin - black collagen - taupe
Ammoniacal silver solution silver nitrate 10% KOH 10%
Gordon & Sweets stain for reticular fibers solutions ferric ammonium sulfate 2.5% (sensitize) formalin (red. silver) ammoniacal silver gold chloride 0.2% sodium thiosulfate potassium permanganate 1% (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 Fixative: Zenker; 10% NBF ok Sections: 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 solution Gram iodine sodium thiosulfate potassium permanganate 0.25% oxalic acid 5%
Mallory PTAH technique for cross-striations & fibrin results cross-striations, fibrin, nuclei - blue collagen - red-brown
PTAH solution hematoxylin phosphotungstic acid water
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 Fixative: 10% NBF; avoid mercury fixatives Sections: 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 0.02% 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 (makes soln. alkaline) silver nitrate Borax (buffer soln.) water
Borax solution sodium borate water
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 water
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 Fixative: 10% NBF, (or post-fix w/) calcium-formalin; avoid alcoholic fixatives Sections: 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 Fixative: 10% NBF Technique: section =< 2mm, stain, process QC: Most tissue has fat
OsO4 paraffin procedure for fat solution OsO4 1% periodic acid 0.5%
OsO4 paraffin procedure for fat results fat - black other elements - 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 purpose Demo mast cells
Toluidine blue for mast cells facts Fixative: 10% NBF QC: section w/ mast cells
Toluidine blue for mast cells solution Toluidine blue water
Toluidine blue for mast cells results mast cells - deep violet background - blue
Plasma cells: Produce immunoglobin, derived from B lymphocytes, MGP
PTAH stains tissue components: Red-brown/salmon, components will lose color if prolonged water or alcohol washing so dehydration after staining must be rapid
If requested blood cells may be demonstrated with? Giemsa
Snook reticulin stain solutions potassium permanganate (ox.) uranyl nitrate (sensitizer) 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. of glycol groups of sugars to aldehydes 2) sensitization 3) silver impregnation 4) reduction of 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 water resorcin dextrin ferric chloride HCl
Created by: CCF