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HT/HTL: Chap8

Connective and Muscle Tissue

Special StainsPurposePrincipleFixative/TechniqueQ.C.Reagents/ProceduresResultsNotes
Aldehyde Fuchsin stains elastic fiber for demonstration of pathologic changes (atrophy of elastic tissue, thinning or loss, reduplication, breaks or splitting from other vascular diesease) or even identify vascular tissues to seen if invaded by tumors HCl and praldehyde added to alcoholic solution of basic fuchsin to form aldehyde fuchsin; will also stain pancreatic beta cell granules and sulfated mucosubstances; staining is intesnsified by prior oxidation NBF preferred; chromates AVOID; Formalin and Bouin -fixed tissues will show colorless bkground; Hg-fixed show pale lilac background; paraffin sections at 4-5u section of aorta embedded on edge or a cross section of a large artery hydrate to 70%R-OH; stain w/ aldehyde fuchsin; rinse w/ 70%R-OH; wash sections in H2O (stops diff); counter w/ LT Grn; dehydrate to xylene ELASTIC FIBERS= deep blue to purple; other tissue elements = Green careful of old stains or old paraldehyde; can use regular aldehyde instead
Russell Modification of MOVAT PENTACHROME STAIN demonstration of MUCIN, FIBRIN, ELASTIC FIBERS, MUSCLE and COLLAGEN acidic muco =alcian blue-monastral fast blue; Fe Hema=elastic fibers, diff by FeCl, sodium thio removes iodine; CROCEIN scarlet and acid fuchsin=stain muscle cytoplas, collagen and ground substan; PTungA diff from collagen, countered by safran NBF or acetic formalin sublimate (HgCL, formaldehyde, DI-water, glaical acetic); paraffin 4-5u Use lung, skin or colon Alcian blue; wash; alkaline ROH; was; Hematoxylin; Rinse; diff in 2% aq Fe3+Cl; rinse; sodium thio; DI-water; rinse in 0.5% acetic acid; wash; croccein scarlet-acid fuchsin; P.T.A.; rinse 0.5% acetic acid; 100% ROH; RoH-safran; dehydrate to xylene NUCLEI and ELASTIC FIBERS = Black; COLLAGEN = Yellow; GROUND SUBSTANCE and MUCIN = Blue; FIBRINOID, FIBRIN = Intense Red; MUSCLE = Red diff of elastic fibers is usally complete in 2-3 min; makesure you remove alkaline ROH -- inhibits rest of staining; CAN DEMONSTRATE CRYPTOCCOCUS NEOFORMANS
Gomori Stain demonstate RETICULIN/RETICULAR FIBERS important in ddx of certain tumors or a change in normal reticular fiber patterns hexose sugars of reticulin oxidezed to aldehydes via KMnO4, excess removed by K-metabisulfite; FeNHSO4 sensitizer, replaced by Ag; formalin reduces Ag to visible; toned w/ AuCl and excess unreduced Ag removed by sodium thio; counter if desired 10%NBF; paraffin 4-5u LIVER tissue oxidize in KMNO; rinse; diff metabsulfate; wash; sensitize in FeNHSO4; wash; impre w/ ammoniacal silver; rinse; reduce formalin; was tone AuCl;rinse;metab; fix in sodium thio; wash/ counter w/ nuclear0fast red; wash well w/ h2o; dehydrate to xylene RETICULIN = Black; COLLAGEN= taupe rinse between diamine silver and formald critical for good retic stain; careful to wash counterstain otherwise can be cloudy; note pattern and make sure the counter does not mask; deposition of silver should be linear not granular!!!
Gordon and Sweets Stain demonstate RETICULIN/RETICULAR FIBERS important in ddx of certain tumors or a change in normal reticular fiber patterns oxidized by KMnO, removed by oxalic acid, ferric ammonio sulfate sensitizer replaced by Ag; formalin reduces Ag; sodium thio removes silver, toned by AuCl; counterstain if desired 10NBF; paraffin at 4-5u LIVER good oxidize w/ KMnO4; rinse; bleach in 1% oxalic acid; was; sensitize in 2.5% Fe NH SO4; wash; impreg w/ Ammoniacal Ag; rinse; reduce in formalin; wash; tone in AuCl; wash; place in sodium thio; rinse; counterstain in nuclear-fast red; dehydrate to xylene REDICULIN = black less background and nuclear staining;
Mallory PTAH demonstrate of muscle cross-striations (diagnostic feature of rhabdomyscaromas or tumors from striated muscle) and fibrin; NEmaline rods present in some skeletal muscle disease mayt also be demonstrated by the method amount of PTA in stain is far greater than hematein (20:1) tungsten binds all available hematein to five blue-colored lake; stains tissue components blue while phosphotungstic acid stain red-brown components; polychromatic! Zenker prefered; 10%NBF ok; cut paraffin at 4-6u skeletal or cardiac muscle for cross-striations, section w/ fibrin for demonstration of fibrin or section of cerebral cortex for glial fibers mordant in Zenkers w/ acetic acid; rinse/ gram iond; rinse/ aq sodium thio/ wwash/ KMnO4; rinse; place in 5% oxalic acid; wash; PTAH overnight; denydrate to xylene CROSS-STRIATIONS, FIBRIN= blue; NUCLEI= blue; COLLAGEN= red-brown replaced by IHC; much better in Zenker; natural ripening is better
Periodic Acid-Methenamine Silver Microwave delineates the glomerular basement membrane Methenamine Ag demonstrates carbohydrate component of basement membranes, oxidezes to aldehydes; Ag ions first bound to carbohydrate components of membrane then reduced; toned; removed 10%NBF; Hg containing fixatives = NO; paraffin at 2u! (KIDNEY) no other control needed for five slieds, use blank slides, use exactly; periodic acid; rinse; slides in coplin w/ methenamine Ag wolution; rinse tone in AuCl; rinse; sodium thio; wash; counter w/ ltgrn;dehydrate to xylene...USE MICROWAVE FOR IMPREG BASEMENT MEMBRANE = black; background = green sharper staining of basement membrane and less background is best w/ microwave; temp is really important; difficult to stain correctly, membrane should really look like a straight line of ink
Oil Red O demonstration pf neutral lipids fat occurring in an abnormal place may be demonstrated; degenerating metrial containing fat such as cell membranes or myelin may coalesce into fat droplets that are demonstrated, tumors from liposarcomas oil-soluble dyes based on greater solubility of dye in the lipod substanses than in the usual hydroalcaholic dye solvents; physical method: 1) dye must be more soluble in tissue lipd; not be water soluble; must be strongly colored; only solution 10%NBF or calcium-formal; NO ALCOHOLIC FIXATIVESl frozen sections at 10u; paraffin cannot be used control is not used cut frozen sections fix in 40% formaldehyde; wash; stain in Oil Red O; wash; stain in Harris Hematoxylin w/ acetic acid; wash/ blueing/ wash/ mount w/ aqueous mounting FAT = intense red; Nuclei = blue NO ALCOHOL; can infiltrate frozen sections w/ sucrose solution before freezing; careful of not squishing fat around;
Sudan Black B in Propylene Glycol neutral lipids; is the most sensitiv eof th elipid dyes more soluton in phospholipids and cerebrosides than oil red O; used in hematophathology to aid in differentiating granulocyte precurosr fro mleukocytes Sudan black B slight baseic and will combine w/ acidic groups in compound lipids thus staining phospholipids; kind of works like oil red O 10% NBF or sections postfixed in calcium-formalin; NO ALCOHOLIC FIXATIVES; frozen sections at 10u; no paraffin sections control not normally used fixed and rinsed frozens in 100% propylene glycol for 15minsx2; stain in SUDAN BLACK B; diff in 85% propylene glycol; wash; counterstain w/ nuclear-fast red; wash FAT = blue-black; nuclei=red same as oil red o
Osmium Tetroxide Praffin Procedure demonstation of fat by a method that allows paraffin embedding of tissue osmium tetroxide chemically combines w/ fat, blackening it in the process; only method fro fat that is chemical; fat combined w/ osmium tetroxide are insoluble in alcohols and xylene and thus paraffin processed 10%NBF; stain west tissue block and then process on the tissue processor bginning in 10% alcohol; no thicker than 2mm or osmium will not penetrate no control used trim 10%NBF tissue to 2mm thick an wash in runnin tap water; place in osmium tetroxide solution; rinse; diff in Periodic acid 0.5%; wash in tap water; process stating at 70%; cute paraffin at 4-5u; deparaffin; stain as H&E or Masson FAT=black; Other tissue elements = according to method used small fat droplets and individual fat cells are demonstrated well not gross amts; face block carefully; cytoplasm will be gray
Toluidine Blue MAST CELLS mAST CELLS STAIN METACHROMATICALLY W/ TOLUIDINE BLUE; STAIN A DIFFERENT COLOR FORM THE DYE AND THE REST OF THE TISSUE; CONTROL SECTION SHOW MAST CELLS STAINED RED-PURPLE; and background stained blue; metachromasia is depended on pH, [dye], and temp 10%NBF; paraffin 4-5u section w/ mast cells hydrate to H2o; stain in toluidine blue; rinse; dehydrate through 95% and absolute alcohol; clear in xylene Mast cells = deep violet; backgound = blue rapid screening method for mast cells, light methelne blue stain works; can be staine dby methyl green-pyronin stain like in nuclear and cytoplasmic staining
Masson Trichrome differentiate between COLLAGEN and SMOOTH MUSCLE in tumors; identify INCREASES in COLLAGENOUS tissue due to disease use of 3 dyes: Biebrich scarlet: all acidophilic tissue element s(cytoplasm, muscle, collagen bind acid dyes); treated w/ phosphotungstic and/or phosphomolybdic (collagen decolored) and linkes aniline blue and pH important too BOUIN preferred, 10%NBF ok; paraffin 4-5u No other tissue needed (internal control); uterus, small intestine, appendix or fallopian ok Mordant in Bouin, heat; wash; WEIGERT; wash; Stain in biebrich scarlet-acid fuchsin; Rinse; phosphomolybdic/phosphothunstic (discard after); stin in ANILINE BLUE; rinse; acetic acid; dehydrate to xylene Nuclei = black; CYTOPLASM, KERATIN, MUSCLE FIBERS = red; COLLAGEN AND MUCUS = blue can use light green; decreased red staining means aged solution, faded blue means overdifferentiated as does burned tissue; picric acid is expolisive; Iron hematoxylis more resistant in subsequent acidic dyes; *CAN microwave, only diff nuclei dark blue
Gomori One-Step Trichrome identify increase in COLLAGENOUS CONNECTIVE TISSUE FIBERS or to differentiate between COLLAGEN AND SMOOTH MUSCLE FIBERS plasma stain (chromotrope 2R) and connective tissue fiber stain (fast-green FCF, light green or aniline blue) combined w/ phosphotungstic acid (favors red staining of muscle and cytoplasm) and glacial acetic acid; tungstate collagen well fixed tissue; Bouin (has picric) is used as mordant; 4-5u No other tissue needed (internal control); uterus, small intestine, appendix or fallopian ok Mordant in Bouin, heat; wash; stain in Weigert; wash; stain in Gomori Trichrome(see principle); diff in 0.5% acetic acid; dehydrate, clear mount Nuclei = Black; CYTOPLASM, KERATIN and MUSCLE FIBERS = Red; COLLAGEN and MUCUS = Green or blue the more acidic a pH the more maximum binding (pH2.5 vs pH1.3), intensity of coloring can be varied
van Gieson Picric Acid-Acid Fuchsin Stain van Gieson excellent counterstain for other methods even if it can be considered a primary connective tissue stain strong acids (low pH), collagen is selectively stained by acid fuchsin (acid aniline dye); picric proveds necessary acidic pH and stains muscle and cytoplasm; the low pH allows for sharpened diff between collagen, cyto and muscle any well-fixed tissue; paraffin 4-5u No other tissue needed (internal control); uterus, small intestine, appendix or fallopian ok Overstain w/ Weigert; wash; stain in van Gieson (discard); 95% alcohol; dehydrate, clear in xylene and mount Nuclei = Black; COLLAGEN = brilliant red; MUSCLE AND CYTOPLASM = Yellow Iron hematox more resistant to acidic dye solutions; do not use water afer van Gieson solution; makes sure picric has enough saturation; usually combined w/ Verhoeff
Verhoeff(-van Gieson) stains elastic fiber for demonstration of pathologic changes (atrophy of elastic tissue, thinning or loss, reduplication, breaks or splitting from other vascular diesease) or even identify vascular tissues to seen if invaded by tumors overstained w/ lake of hematoxylin-ferric chloride-iodine converting hematox to hematein-REGRESSIVE; diff'd by excess mordant, ferric chloride, elastic tissue has strongest affinity for the lake so other tissue elements decolor; sodium thio removes excess well-fixed tissue, NBF or Zenker preferred; paraffin 4-5u section of aorta embedded on edge or a cross section of a large artery Verhoeff stain(alcholic hemat, ferric chloride, lugol blue), wash; diff in 2% ferric chloride background is colorless; rinse; sodium thosulfate; wash; ccounter in van Gieson; diff in 95% ROH; dehydrate to xylene ELASTIC FIBERS = blue-black to black; nuclei = blue to black; COLLAGEN = RED; other tissue elements = yellow Van Gieson most commonly used counterstain usually a/ van Gieson; careful of overdifferentiating stain; don't treat w/ alcohol and you can restain; remove Hg deposits before staining; van Gieson must be right pH;
Created by: Miellee