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HT/HTL: Chap11

Pigments, Minerals, and Cytoplasmic Granules

SPECIAL STAINPURPOSEPRINCIPLEFIXATIVE/TECHNIQUEQUALITY CONTROLREAGENTS/PROCEDURERESULTSNOTES
TURNBULL BLUE detects ferrous (Fe2+) in tissues Fe NOT stored in tissue, Fe2+ is toxic Ferrous iron + potassium ferricyanide --HCl--> Turnbull blue (ferrous ferricyanide) Alcohol or 10%NBF, 4-5 microns Section w/ Ferrous Iron 1) FRESH ferricyanide solution, 1 hr 2) wash in 1% Acetic Acid 3)Counter w/ nuclear-fast red 4) Rinse in DI H2O , dehydrate, clear (xyl) Fe2+ = blue Background = pink/red --used in Schmorl
SCHMORL indication of REDUCING SUBSTANCES MELANIN, ARGENTAFFIN GRANULES, FORMALIN PIGMENT will stain reducing substance + ferric iron (Fe3+) --> Ferrous Iron (Fe3+) + ferricyanide --> Turnbull Blue 10%NBF preferred, 4-5 microns w/ melanin or argentaffin granules 1) slides in ferric chloride-potassium cyanide working solution 2) rinse in h2o 3) counter w/ metanil yellow 4) rinse in h20 4)dehydrate, clear, mount Reducing substances = blue green Goblet cells, mucin = rose Background = yellow-green GI tract good (mucin demonstrated), careful of glassware and FRESH solutions
FONTANA-MASSON demonstrate argentaffin substance s(melanin, granules of carcinoid tumors, some neurosecretory granules) ARGENTAFFIN = ability to bind silver from a silver solution and reduce it to visible Ag w/o need of separate reducing agent 10%NBF, NO ALCOHOL -- dissolves argentaffin granules, 4-5 microns Skin w/ melanan; small intestine or appendix for argentaffin granules 1)Slides in silver nitrate solution w/ heat; PROGRESSIVE look for dark brown granules and colorless background 2) rinse in h20 3) gold chloride toner 3) rinse 4) sodium thiosulfate 5) rinse 6) counter w/ nuclear-fast red 7) wash, dehydrate, clear Melanin = black Argentaffin = black Nuclei = Pink -not specific for either melani or argentaffin -use silver only once -OVERSTAINING = DIRTY GRAY BAVKGROUND AND LOSS OF CONTRAST
MICROWAVE FONTANA-MASSON see fontana masson see fontana masson see fontana masson see fontana masson see fontana masson Argentaffin cell granules, chromaffin granules, melanin and other argentaffin substances = black nuclei - pink bleach melanin for a negative control slide?
GRIMELIUS ARGYROPHIL STAIN demonstrate argyrophil granules in neurosecretory tumors ARGYROPHIL, 10%NBF, 4-5 microns argyrophil-positive carcinoid tumor is preferred; can use small intestine 1) incubate in silver nitrate 2) reduce (hydroquinone, sodium sulfite, h2o) 3)rinse, repeat silver nitrate then reducing solution 4) rinse and counter w/ nuclear fast red Argentaffin, argyrophil = dark brown to black nuclei = red background = pale yellow-brown should compare w/ fontana-masson results to see comparison of staining of argentaffin granules
CHURUKIAN-SCHENK demonstrate aregyrophil granules in neurosecretory tumors argyrophils 10%NBF, 4-5 microns argyrophil positive carcinoid tumor, small intestine works 1) (hydrate to acidified h2o/citric acid-glycine,) incubate in silver nitrate, rinse 2)transfer to reducing solution (hydroquinone, sodium sulfate) 3)rinse, repeat 1&2 4)Rinse, counter w/ nuclear-fast red 5) rinse, dehydrate, clear, mount argyrophil & argentaffin = black* nuclei = red (*orange to red) background = yellow-brown (*light yellow-orange) *can microwave
GOMORI-METHENAMINE-SILVER (GMS) demonstrate urates (GOUT) around joints and soft tissues, collection of the crystals = gouty tophi Urates stained black via silver, which is then reduced to metallic form ABSOLUTE R-OH, 4-5 microns section w/ urates 1)from absolute alcohol, place in methenamine silver solution to incubate until crystals are black 2) rinse, place in 3% sodium thiosulfate 3) rinse, counter w/ light green 4)dehydrate, clear, mount URATES = black background = green Ca2+ can be demonstrated if deposits are large
BILE STAIN demonstrate bilirubin in tissue vilirubin is oxidized to biliverdin, green color develops, in an acid medium using ferric chloride in trichloracetic acid medium 10%NBF, 4-5 microns, may use frozen sections tissue w/ bile 1) Fouchet reagent (trichloracetic acid, ferric chloride, 20%) 2) rinse, stain w/ van Gieson solution (acid fuchsin and saturated picric) 3)differentiate in 95% etoh 4)rinse, dehydrate, clear, mount bile/bilirubin = emerald green to olive drab background = yellow
VON KOSSA presence of calcium in tissue CaCO3 + 2AgNO3 --> Ag2CO3 + Ca(NO3)2 Ag2CO3 + sunlight reduction --> Metallic silver, co2 and h2o alcohols preferred or 10%NBF section w/ calcium 1) distilled h2o, silver nitrate, expose to sunlinght. progressive so stock when calcium salts are brown-black 2) rinse, plase in sodium thosulfate 3) rinse, counter w/ nuclear red 4) wash, dehydrate, clear and mount Calcium salts = black background red detects anions combined w/ calcium NOT calcium
ALIZARIN RED S detects calcium in tissue react w/ cations calcium, magnesium, manganes, barium and strontium...forms an alizarin red S-calcium complex in a CHELATION process alcholic formalin, or 10%NBF, 4-5 microns Section w/ calcium place in alizarin red s staning solution (2%, ph critical to 4.1-4.3) place 2) shake of dye, blot sections 3) dehydrate, clear, mount Calcium deposits = orange red, birefringent like Dahl
RHODANINE detection of Cu, especially in liver of Wilson disease SENSITIVE, demonstrates protein to which copper binds rather than the copper itself can get false-positives 10%NBF, 6-8 microns section w/ copper 1) place slides in working rhodanine solution for 18hours at body temp 2)rinse, stain in Mayer 3) rinse, quick in sodium borate solution 4) rinse, dehydrate, clear, mount Copper = bright red to red yellow nuclei = light blue need to have high copper concentration otherwise will fad, do not overstain w/ hematoxylin, can mask copper *can microwave
PRUSSIAN BLUE detects LOOSELY bound ferric (Fe3+) normal: small amounts in bone marrow and spleen abnormal: LARGE deposists in hemochromatosis and hemosiderosis 3K4Fe(CN)6 + 4Fe3+ --HCL--> Prussian blue pigment + 12K+ Alcohol or 10%NBF 4-5 microns Section w/ Ferric iron, but not w/ excessive amounts of it--rxn product is slightly soluble and may contaminate during incubation 1)equal 2% Potassium Ferrocyanide & 2% HCL, Heat 2)Wash in DI H2O 3)Counter w/ NUCLEAR-Fast Red 4)Rinse in tap, dehydrate, xylene Nuclei/Hemofuschin = bright red Hemosiderin (Fe) = Blue Background = Pink -stain jar is chemically clean; can result in diffuse background staining -make sure to rinse excess nuclear-fast red counterstain otherwise cloudy slides result
Created by: Miellee