Special Stain Final

DEPARTMENT OF PATHOLOGYSBKS MEDICAL INSTITUTE& RESEARCH CENTRE

PIPARIA

ALCIAN BLUE pH2.5 - ACID MUCOPOLYSACCHARIDES

PURPOSE: Alcian blue stains acid mucosubstances and acetic mucins.Excessive amounts of non-sulfated acidic mucosubstances are seen inmesotheliomas, certain amounts occur normally in blood vessel walls butincrease in early lesions of atherosclerosis.

PRINCIPLE: Alcian blue is a group of polyvalent basic dyes that are watersoluble. The blue color is due to the presence of copper in the molecule.The 3% acetic acid solution (pH2.5), Alcian blue stains both sulfated andcarboxylated acid mucopolysaccharides and sulfated and carboxylatedsialomucins (glycoproteins). It is believed to form salt linkages with theacid groups of acid mucopolysaccharides.

CONTROL: Small intestine, appendix, or colon.

FIXATIVE: 10% NBF, Bouin’s, or Hollande's.

TECHNIQUE: 4m paraffin sections.

EQUIPMENT: Rinse glassware in DI water. Coplin jars, pH meter,

REAGENTS:

3% Glacial Acetic AcidAcetic acid 3.0 mlDistilled water 100.0 mlSolution is stable for 1 year.CAUTION: Contains acid.

Alcian Blue Solution:3% glacial acetic acid 100.0 mlAlcian blue 8GX 1.0 gmMix, adjust pH to 2.5, using aceticacid. Filter, add a crystal ofthymol, label with initial and date.Solution is stable for 2 to 6months.CAUTION: Contains acid, avoid contactand inhalation of dye.

Nuclear Fast Red (Kernechtrot):Aluminum sulfate 25.0 gmDistilled water 500.0 mlNuclear fast red 0.5 gmDissolve the aluminum sulfate inthe water. Add the nuclear fast red,dissolve with aid of heat. Filter,add a crystal of thymol. Stable for1 year.CAUTION: IRRITANT avoid contact andinhalation

CARBOHYDRATESALCIAN BLUEPROCEDURE:1. Hydrate slides to distilled water.2. 3% acetic acid, 3 minutes.3. *Alcian blue solution, 30 min.4. Wash in running water for 2 minutes, rinse in distilled.5. Nuclear-fast red, 5 minutes, wash in tap water.6. Dehydrate, clear, and coverslip.

RESULTS:Acid mucins/mucosubstances: blueNuclei (using Nuclear fast red) reddish pink


PIPARIA

IRON - PRUSSIAN BLUE REACTION - MALLORY'S METHOD

PURPOSE: To demonstrate ferric iron in tissue sections. Small amountsof iron are found normally in spleen and bone marrow. Excessive amountsare present in hemochromatosis, with deposits found in the liver andpancreas, hemosiderosis, with deposits in the liver, spleen, and lymphnodes.

PRINCIPLE: The reaction occurs with the treatment of sections in acidsolutions of ferrocyanides. Any ferric ion (+3) in the tissue combines withthe ferrocyanide and results in the formation of a bright blue pigmentcalled 'Prussian blue" or ferric ferrocyanide.

CONTROL: A known positive control tissue.

FIXATIVE: 10% formalinTECHNIQUE: Cut paraffin sections 4μ.EQUIPMENT: Microwave oven, acid-cleaned glassware, non-metalicforceps.

REAGENTS:5% Potassium Ferrocyanide:Potassium ferrocyanide 25.0 gmDistilled water 500.0 mlMix well, pour into an acid-cleanedbrown bottle. Stable for 6 months.CAUTION: Low toxicity if not heated..

Nuclear-fast Red:

5% Hydrochloric Acid:Hydrochloric acid, conc. 25.0 mlDistilled water 475.0 mlMix well, pour into brown bottle,stable for 6 months.CAUTION: Corrosive, avoid contact andinhalation.

Working Solution:5% potassium ferrocyanide 25.0 ml5% hydrochloric acid 25.0 mlMake fresh, discard after use..PROCEDURE:1. Deparaffinize and hydrate to distilled water.2. *Working solution, * microwave, 30 seconds. Allow slides to stand insolution for 5 minutes, in the fume hood.3. Rinse in distilled water.4. Nuclear-fast red, 5 minutes.5. Wash in tap water.6. Dehydrate, clear, and coverslip.*Conventional method: room temperature for 30 minutes.RESULTS:Iron (hemosiderin) blueNuclei redBackground pink


PIPARIA

FITE'S ACID FAST STAIN - LEPROSY

PURPOSE: To demonstrate mycobacterium leprae (leprosy), which are acidfast organisms.PRINCIPLE: This technique combines peanut oil with the deparaffinizingsolvent (xylene), minimizing the exposure of the bacteria's cell wall toorganic solvents, thus protecting the precarious acid-fastness of theorganism.CONTROL: Leprosy positive tissue.FIXATIVE: 10% formalinTECHNIQUE: Cut paraffin sections 4-5 microns.EQUIPMENT: Rinse all glassware in DI water, coplin jars, bibulousblotting paper.REAGENTS:Xylene/Peanut Oil Solution:Xylene 50.0 mlPeanut Oil 50.0 mlMix well. Label with date andinitials, solution is stable for 1year.CAUTION: Flammable, irritant.Ziehl-Neelsen Carbol-Fuchsin:

1% Acid Alcohol:

Working Methylene Blue:FITE'S Page: 2 of 2Hydrochloric acid: strong irritant to skin, eyes and respiratory system.

Target organ effects via inhalation on skin, respiratory, reproductive andfetal systems.Methylene blue: produced deleterious effects on fertility in rats.

PROCEDURE:1. Deparaffinize in xylene/peanutoil mixture, 2 changes, 10minutes each.2. Drain slides, blot off excessoil.3. Rinse in distilled water untilslide clears.4. Carbol-fuchsin, 30 minutes,room temperature.5. Wash in tap water.6. Acid alcohol until pale pink, dipuntil stain stops running.7. Wash in tap water.8. Counterstain in WorkingMethylene blue, 30 seconds.9. Wash in tap water.10. Blot and air dry.11. Dip in xylene and coverslip.

RESULTS:Acid-fast bacilli redBackground blueNOTE: Mineral oil may be substituted for peanut oil.PROCEDURE CARD


PIPARIA

RETIC - GORDON AND SWEET'S METHOD - RETICULAR FIBERS

PURPOSE: A silver impregnation technique that demonstrates reticularfibers. Reticulum is a support function of the body and is abundant inliver, spleen, and kidney. In a normal liver the fibers are will definedstrands, but necrotic and cirrhotic liver show discontinuous patterns.Reticulum also forms characteristic patterns in relationship to certaintumor cells.

PRINCIPLE: The tissue is oxidized, then sensitized with the iron alum,which is replaced with silver. The silver is reduced with formalin to itsvisible metallic state.

CONTROL: Normal liver.

FIXATIVE: 10% formalin

TECHNIQUE: Cut paraffin sections at 4m to 5m.

EQUIPMENT: Acid cleaned glassware, pipettes.

REAGENTS:3% Sulfuric Acid:Distilled water 100.0 mlSulfuric acid 3.0 mlMix well, label with initial anddate. Solution is stable for 1 year.Potassium Permanganate:Potassium permanganate 0.5 gmDistilled water 47.5 ml3% Sulfuric acid 2.5 mlMake fresh, discard after use.1% Oxalic Acid:Oxalic acid 1.0 gmDistilled water 100.0 mlMix well, label with initial anddate. Solution is stable for 1 year.

2% Iron AlumFerric ammonium sulfate 2.0 gmDistilled water 100.0 mlMix well, label with date andinitial. Solution is stable for 6months.

10% Silver Nitrate StockSilver nitrate 10.0 gmDistilled water 100.0 mlAcid clean all glassware and rinsewell. Store in a brown bottle, keepin the refrigerator. Label withinitial and date. Stable for 6months.

3% Sodium Hydroxide Stock:Sodium hydroxide 1.5 gmDistilled water 50.0 mlMix well, label with date andinitials. Store in the refrigerator,stable for 3 months.

Ammoniacal SilverWorking Solution:10% silver nitrate 5.0 mlAdd ammonium hydroxide drop bydrop until clear again.3% sodium hydroxide 5.0 mlAdd ammonium hydroxide drop bydrop until clear again.Distilled water 40.0 mlUsing acid clean glassware, agitatesolution continually while addingammonium hydroxide. Make rightbefore use, discard.

10% Formaldehyde:Formaldehyde 5.0 mlDistilled water 45.0 mlMake fresh, discard after use.

0.5% Gold Chloride:Gold chloride 1.0 gm


PIPARIA

Distilled water 200.0 mlUse acid clean glassware, labelwith date and initials. Store in therefrigerator. Stable for 1 year.5% Hypo

Nuclear-Fast Red (Kernechtrot)

PROCEDURE:1. Deparaffinize and hydrate to distilled water.2. Potassium permanganate solution, 5 minutes.3. Wash in water.4. 5% oxalic acid until clear.5. Wash in distilled water.6. Iron alum solution, 10 minutes.7. Wash in running tap water, rinse in distilled, 3 changes.

8. Silver solution, 7 dips, shake excess solution off slides.9. Distilled water, 2 changes, 3 quick dips each.10. 10% formaldehyde solution until gray black, 30 seconds.11. Wash in distilled water.12. 0.5% Gold chloride, 1 minute.13. Rinse in distilled water.14. 5% hypo, 1 minute.15. Wash in tap water.16. Nuclear-fast red solution, 5 minutes.17. Wash in running tap water.18. Dehydrate, clear, and coverslip.

RESULTS:Reticular fibers blackNuclei red5. Change distilled water after every


PIPARIA

LIPOFUSCIN - SUDAN BLACK BPURPOSE: Lipofuscins, the "wear-and-tear" pigment, collects in the morepermanent cells (heart, liver, and neurons) of older persons. Lipofuscin isthe accumulation of lysosomes, which have absorbed the worn-out,undigestible parts of the cell and known as residual bodies. It is a yellowbrownpigment, and will stain after being processed in paraffin.

PRINCIPLE: The lipofuscins react with oil-soluble dyes.

CONTROL: Bielschowsky control tissue, cut at 5μ.

FIXATIVE: 10% formalin

TECHNIQUE: Cut paraffin sections 5μ.REAGENTS:Sudan Black/Lipofuscin:Sudan Black B saturated70% alcohol 50.0 ml

Mix well, filter through filterpaper, then filter again through afrittered glass filter of mediumporosity with suction. Stable for 6months.

PROCEDURE:1. Deparaffinize and hydrate slides to 70% alcohol.2. Sudan black, overnight, room temperature.3. Rinse and differentiate in 70% alcohol until background is pale gray.4. Wash will in tap water.5. Mount in aqueous mountant.MINERALS AND PIGMENTS

LIPOFUSCIN - SUDAN BLACK B

RESULTS:Lipofuscins, RBC's: black


PIPARIA

PAPANICOLAOU STAINING PROCEDURE

Intended Use:For staining exfoliated cells in cytological specimens.Summary and Explanation:The Papanicolaou Staining procedure is used for examining exfoliated cells in secretions,exudates, transudates or scrapings of various internal organs and tissue. Cells are fixedto a slide and stained first with Hematoxylin, which stains the nuclei followed by OG-6and EA-50 or EA-65 as a counterstain.Reagents:EA-50 Multiple Polychrome Stain1000mL Catalogue # IMEA50Light Green S.F. Yellowish, Fast Green FCF, Bismark Brown Y, Eosin Y,Phosphotungstic Acid, Glacial Acetic Acid. Filter before use.EA-65 Multiple Polychrome Stain1000mL Catalogue # IMEA65Light Green S.F. Yellowish, Fast Green FCF, Bismark Brown Eosin Y, PhosphotungsticAcid, Glacial Acetic Acid. Filter before use.OG-6 Orange G Stain1000mL Catalogue # IMOG6Orange G, Phosphotungstic Acid. Filter before use.Harris Hematoxylin1000mL Catalogue # IMHARRISHXHematoxylin, Potassium Alum, Glacial Acetic Acid, Sodium Iodate. Filter before use.EA-50 and OG-6 were developed as general stains for vaginal smears. EA-65, amodification of EA-50, has been especially developed for use with OG-6 in the study ofsmears from urinary, paracentetic, thoracentetic material, from sputum, from gastric andexternal ulcerated sources as well as those smears which, are heavily mixed with mucus.In such smears EA-65 helps retain the translucency essential to proper cytological study.EA-65 is interchangeable with EA-50 in the staining procedure. Staining times andtechnique remain the same.Storage and Stability:Store at 15°-30°C. Reagents are stable until expiration dates shown on the label.Warnings and Precautions:Papanicolaou Staining reagents are flammable and toxic. Keep away from sources ofignition. In case of contact with eyes, rinse immediately with water and seek medicaladvice. May be fatal or cause blindness if swallowed.Dispose of waste in accordance with applicable laws.Materials Required But Not Provided:Ethanol CoverslipsHydrochloric Acid MicroscopeAmmonium Hydroxide Microscope slidesXylene MethanolPage 4Specimen Collection:In the collection and preparation of smears for cytological examination, the majorobjectives are:

1. Specimens should have a sufficient number of cells from the area in question.2. Smears should contain well preserved cells uniformly distributed so that eachcell can be individually examined.3. The staining procedure should clearly define the details of all structures.Cytological preparations are obtained from patient by approved methods and techniques.Scraping, obtained from the vagina, uterus, cervix, mouth or ulcerated skin area is spreaddirectly on a clean microscope slide.Sediment (obtained by centrifugation or filtration) from bodily secretions is spread on aclean microscope slide.The smear is immediately fixed with a cytological spray fixative or in an alcohol-ether dip.Fixation or preservation is one of the most important steps in the procedure. Drying of thecells prior to fixation will usually result in artifacts such as nuclear distortion andvacuolization.After fixation there are no special handling requirements for cytological smears. However,smears which are to be mailed to a laboratory, should remain in the fixative for about onehour. A second clean glass slide may be placed on each fixed slide for protection.Procedure:Notes:Filter the Harris Hematoxylin immediately before use.1. Dip slide(s) gently 5-10 times in 95% ethanol.2. Dip slide(s) gently 5-10 times in 70% ethanol.3. Dip slide(s) gently 5-10 times in distilled water.4. Stain 5 minutes in Harris Hematoxylin.5. Place smears in distilled water. Rinse in successive changes of distilled wateruntil the water remains colourless.6. Dip slide(s) gently 5-10 times in 70% ethanol.7. Dip slide(s) in a 1% solution of HCl in 70% ethanol until the smear shows asalmon colour.8. Rinse slide(s) well in 2 changes of 70% ethanol.9. Dip slide(s) gently in a 3% solution of ammonium hydroxide in 70% ethanoluntil the smear takes on a blue colour.10. Rinse the slide(s) in two changes of 70% ethanol.11. Dip slide(s) 5-10 times in 95% ethanol.12. Stain slide(s) in OG-6 for 2 minutes.13. Rinse slide(s) in two changes of 95% ethanol.14. Stain slide(s) in EA-50 or EA-65 for 3-6 minutes.15. Rinse slide(s) well in two changes of 100% methanol.16. Rinse slide(s) in one part absolute methanol one part xylene.17. Clean smear in xylene.Page 2 Page 3Mounting Procedure:1. After the smear has been completely cleaned in xylene it is mounted with amicroscope slide cover glass preferably 22x40mm, #1 thinness.2. A permanent clean mounting medium should be used.3. The excess xylene should be drained, in order to avoid the appearance of airspaces when xylene evaporates.


PIPARIA

4. Place the required amount of mounting medium along an edge of one of thelonger borders of the coverslip.5. Place the slide at right angles to the edge of the coverslip so that the sidecontaining the cells is facing the mounting medium.6. Slowly lower the slide and permit the mounting medium to spread between theslide and coverslip.Results:Nuclei are stained blue while cytoplasm displays varying shades of pink, orange, yellowand green.Limitations:1. Proper specimen collection and fixation of cells is essential for interpretation.

References:1. Papanicolaou GN: Atlas of Exfoliative Cytology, Harvard University Press,Cambridge, 1954.2. United States, Naval Medical School, National Naval Medical Center, Bethesda,Maryland, Manual of gynecological exfoliative cytology. US Government PrintingOffice, Washington, D.C., 1965.3. A Manual for Cytotechnology, C. Keebler, J. Reagen, Editors, American Societyof Clinical Pathologists Press, Chicago (IL), 1983.


PIPARIA

Hematoxylin and Eosin (H&E) staining

•Place slides containing paraffin sections in a slide holder (glass or metal)•Deparaffinize and rehydrate sections:3 x 3´ Xylene (blot excess xylene before going into ethanol)3 x 3´ 100% ethanol1 x 3´ 95% ethanol1 x 3´ 80% ethanol1 x 5´ deionized H2O•While sections are in water, skim surface of hematoxalin with a Kimwipe to remove oxidized particles. Blot excess water from slide holder before going into hematoxalin.•Hematoxalin staining:1 x 3´ HematoxalinRinse deionized water1 x 5´ Tap water (to allow stain to develop)Dip 8-12x (fast) Acid ethanol (to destain)Rinse 2 x 1´ Tap waterRinse 1 x 2´ Deionized water (can leave overnight at this stage)•Blot excess water from slide holder before going into eosin.•Eosin staining and dehydration:1 x 30 sec Eosin (up to 45 seconds for an older batch of eosin)3 x 5´ 95% ethanol3 x 5´ 100% ethanol (blot excess ethanol before going into xylene)3 x 15´ Xylene

•You can leave slides in xylene overnight to get good clearing of any water.•Coverslip slides using Permount (xylene based).•Place a drop of Permount on the slide using a glass rod, taking care to leaveno bubbles.•Angle the coverslip and let fall gently onto the slide. Allow the Permount tospread beneath the coverslip, covering all the tissue.•Dry overnight in the hood.

Reagents for H&E staining:• Xylene: StatLab (Lewisville, TX) #8400Laboratory grade, Anapath brand• Acid Ethanol: 1 ml concentrated HCl + 400 ml 70% ethanol• Hematoxylin: Poly Scientific (Bayshore, NY) #s212AHarris hematoxylin with glacial acetic acid• Eosin: Poly Scientific (Bayshore, NY) #s176Eosin Phloxine stain, working• Permount: Fisher Scientific #SP15-100Histological mounting medium


PIPARIA

MASSON TRICHROME STAINING(Michelle's protocol, 9/01)

REAGENTS NEEDED:• Sigma Accustain Trichrome Stain Kit (Catalog #HT15) contains:Biebrich Scarlet-Acid Fuchsin Solution (# HT15-1, 0.9% biebrich scarlet, 0.1% acid fuchsin, 1%acetic acid), Phosphotungstic Acid Solution (#HT15-2, 10% phosphotungstic acid),Phosphomolybdic Acid Solution (#HT15-3, 10% phosphomolybdic acid ), andAniline Blue Solution (#HT15-4, 2.4% aniline blue, 2% acetic acid)• Bouin's Solution (Sigma Catalog #HT10132-1L or HT101128-4L)• Weigert's Iron Hematoxylin Set (Sigma catalog #HT10-79)1. Deparaffinize slides and rehydrate sections:3 x 3´ Xylene (blot excess xylene before going into ethanol)3 x 3´ 100% ethanol1 x 3´ 95% ethanol1 x 3´ 80% ethanol1 x 5´ deionized H2O2. Mordant in Bouin's Solution at room temperature overnight in a hood. Be careful, Bouin'ssolution is hazardous and the picric acid, when in less than 10% water, is veryexplosive. Used Bouin's solution should be placed in an appropriate waste container.** Bouin's Solution intensifies the final coloration of the tissue.3. Wash slides in running tap water to remove yellow color from sections. Rinse briefly indistilled water.4. Stain in Working Weigert's Iron Hematoxylin Solution for 5 minutes. Make Hematoxylin

Solution fresh by adding equal volumes of Solution A (1% Hematoxylin in 95% EtOH) andSolution B (1.2% Ferric Chloride and 1% Acetic Acid in distilled water). The working solutionis good for approximately 10 days.** Hematoxylin stains nuclei blue-black.5. Wash in running tap water for 5 minutes. Rinse in deionized water.6. Stain in Biebrich Scarlet-Acid Fuchsin for 5 minutes.Decreased red staining usually indicates that the staining solution has aged or beenoverused and should be discarded.** Beibrich scarlet-acid fuchsin stains cytoplasm and muscle red.7. Rinse in deionized/distilled water.8. Place the slides in Phosphomolybdic/Phosphotungstic Acid Solution for 5-10 minutes.Freshly prepare Working Phosphotungstic/Phosphomolybdic Acid Solution by mixing 1volume of Phosphotungstic Acid Solution and 1 volume of Phosphomolybdic Acid Solutionwith 2 volumes of distilled water. Discard after one use. Formation of a precipitate inPhosphomolybdic Acid Solution does not affect performance.** This allows for uptake of the aniline blue stain.9. Stain sections in Aniline Blue Solution for 5 minutes.


PIPARIA

**Aniline blue stains collagen blue.10. Rinse slides briefly in distilled water.

11. Place slides in 1% acetic acid solution for 3-5 minutes. Discard this solution.

MASSON TRICHROME STAINING

** Rinsing in acetic acid after staining renders the shades of color more delicate andtransparent.** If blue staining of connective tissue appears faded, the section has probably beenoverdifferentiated in the acetic acid solution.12. Dehydrate to xylene.2 x 3´ 95% ethanol2 x 3´ 100% ethanol (blot excess ethanol before going into xylene)3 x 5´ Xylene

13. Leave slides in xylene overnight to get good clearing of the ethanol.14. Coverslip slides using Permount or Polymount (xylene based).•Place a drop of Permount on the slide using a glass rod.•Angle the coverslip and let fall gently onto the slide.•Allow the Permount to spread beneath the coverslip, covering all the tissue.•Dry overnight in the hood.


PIPARIA

ALCIAN BLUE-PAS (PAB)

PURPOSE: To differentiate between neutral and acetic mucosubstances.Routine stain for G.I. biopsies.CONTROL: Use a mucin control.FIXATIVE: 10% formalin or Hollande's fixative.TECHNIQUE: Cut paraffin sections 4-5 microns thick.EQUIPMENT: Rinse glassware in DI water. Coplin jar, microwave oven.REAGENTS:Alcian Blue, pH2.5:See Alcian Blue0.5% Periodic Acid:See PASSchiff's Reagent:See PASHematoxylin Gill-3:Purchased through BaxterSAFETY: Wear gloves, goggles, particle mask and lab coat, avoid contactand inhalation. Work under the hood, keep hot, uncapped, solutions underthe hood.Schiff’s Reagent: Use extreme caution, Basic fuchsin (pararosaniline) is aknown carcinogen.PROCEDURE:1. Deparaffinize and hydrate to distilled water.2. *Alcian blue pH2.5, microwave Hi power, 45 seconds, let stand for 2-5 minutes.3. Wash in running tap water, 5 minutes, rinse in distilled.4. 0.5% Periodic acid, 5 minutes.5. Rinse in distilled water.6. *Schiff's reagent, microwave Hi power, 45 seconds, allow to stand for

2-5 minutes.7. Wash in running tap water for 5 minutes, rinse in distilled.CARBOHYDRATESALCIAN BLUE-PAS Page: 2 of 28. Hematoxylin, 1-3 minutes, wash in water.9. Acid rinse, 10 dips, wash, bluing solution, 10 dips.10. Wash in running tap water for 5 minutes.11. Dehydrate, clear, and coverslip.*Conventional Method: Alcian blue, 30 minutes, room temperature.*Schiff's reagent, 30 minutes, room temperature.RESULTS:Acid mucosubstances blueNeutral polysaccharides magentaREFERENCES:Sheehan D, Hrapchak B, Theory and practice of Histotechnology, 2nd Ed,1980, pp 163,173-174, Battelle Press, OhioBancroft J, Stevens A, Theory and Practice of Histological Techniques, 2ndEd, 1982,pp194-98,Churchill Livingstone, NY.Carson F, Histotechnology A Self-Instructional Text, 1st Ed, 1990,pp130-131, ASCP, IllCrookham, J, Dapson, R, Hazardous Chemicals in the HistopathologyLaboratory, 2nd ED, 1991, Anatech LTD


PIPARIA

PAS - McMANNUS' PERIODIC ACID SCHIFF'S - GLYCOGEN

PURPOSE: Glycogen is present in skin, liver, parathyroid glands andskeletal and cardiac muscle. The PAS stain is used for demonstration ofbasement membranes, fungus secreting adenocarcinoma fromundifferentiated squamous cell carcinoma, and mucosubstances secretedfrom the epithelia of various organs. A routine stain for liver and kidneybiopsies.PRINCIPLE: The PAS stain is a histochemical reaction in that theperiodic acid oxidizes the carbon to carbon bond forming aldehydes whichreact to the fuchsin-sulfurous acid which form the magenta color.CONTROL: For staining fungus; use a known positive such as those usedfor the GMS. Use skin, aorta or normal liver for positive PAS staining.FIXATIVE: Any well fixed tissue.TECHNIQUE: Cut paraffin sections 4-5 m( 3m for kidney biopsies).EQUIPMENT: Rinse glassware in DI water. Coplin jar, microwave oven.REAGENTS:0.5% Periodic Acid:Periodic acid 0.5 gmDistilled water 100.0 mlMix well, label with date andinitial. Stable for 1 year.Caution: Avoid contact and inhalation.Hematoxylin, GILL-3 Purchasedthrough Baxter.CARBOHYDRATESPAS STAIN Page: 2 of 3Lillie's Cold Schiff's Reagent:Basic fuchsin 10.0 gmSodium metabisulfite 18.0 gmDistilled water 1000 0 ml

Hydrochloric acid 10.0 mlStir solution for 2 hours, set in adark cool place overnight. Thesolution is now a clear light brownto yellow color. Add:Activated charcoal 500.0 gm(or two heaping spoons)Stir. Filter through Whatman #2filter paper into a 1000 mlgraduated cylinder. Change thefilter paper often. Restore volumeto 1000 ml with distilled water.Store in Refrigerator, solution isstable for 6 months.CAUTION: Carcinogen, corrosive.SAFETY: Wear gloves, goggles and lab coat. Avoid contact and inhalation.Hydrochloric acid: strong irritant to skin, eyes and respiratory system.Target organ effects via inhalation on skin, respiratory, reproductive andfetal systems. Corrosive.Schiff’s Reagent: Use extreme caution, Basic fuchsin (pararosaniline) is aknown carcinogen. Wear gloves, goggles, particle mask and lab coat, whilepreparing solution. Work under the hood, keep hot, uncapped, solutionsunder the hood.CARBOHYDRATESPAS STAIN Page: 3 of 3PROCEDURE:1. Deparaffinize and hydrate to distilled water.2. Place slides into 0.5% Periodic acid for 5 minutes.3. Rinse in distilled water.4. *Schiff's Reagent, microwave HIGH power, for 45 - 60 seconds, untildeep magenta.


PIPARIA

PAS - McMANNUS' PERIODIC ACID SCHIFF'S - GLYCOGEN

5. Wash in running tap water for 5 minutes.6. Counterstain in hematoxylin for 3 minutes.7. Wash in tap water, blue hematoxylin, rinse in distilled water.8. Dehydrate in alcohol, clear, and coverslip.* Conventional method: Schiff's Reagent, room temperature for 30minutes.RESULTS:Glycogen, fungus: magentaNuclei blueNOTES:1. To check stain, pour 10 ml of formaldehyde in cylinder, add a fewdrops of Schiff's, it should turn red-purple immediately.

2. Discard Schiff's if it turns pink while sitting in the refrigerator.3. The white precipitate at the bottom of the Schiff's can be redissolvedif desired by gently warming and stirring the solution.REFERENCES:Sheehan D, Hrapchak B, Theory and practice of Histotechnology, 2nd Ed,1980, pp 164-166, Battelle Press, OhioBancroft J, Stevens A, Theory and Practice of Histological Techniques, 2ndEd, 1982, pp 188-190, Churchill Livingstone, NYCrookham, J, Dapson, R, Hazardous Chemicals in the HistopathologyLaboratory, 2nd ED,1991, Anatech


PIPARIA

PAS/D - GLYCOGEN DIGESTION - DIASTASE

PURPOSE: To determine glycogen by digesting out and staining with PASstain.PRINCIPLE: The diastase (or a-amylase) act on glycogen to de polymerizeit into smaller sugar units, maltose and glucose, that are washed out ofthe section.CONTROL: Identical sections are obtained on two separate slides. One isdigested the other is not, both are stained with the PAS stain. Liver is agood control.FIXATIVE: Any well fixed tissue.TECHNIQUE: Cut paraffin sections at 4m to 5mEQUIPMENT: Rinse glassware in DI water. Coplin jars, 37°C oven.REAGENTS:Digestion Solution:Diastase (with a-amylase)0.05 gm(obtained through Ameri. Reagent)Distilled water 50.0 mlMake fresh, use within 15 minutes,discard.0.5% Periodic Acid:See PASSchiff's Reagent:See PASHematoxylin, Gill-3Purchased through BaxterSAFETY: Hydrochloric acid is caustic use caution, flush with water. Weargloves, goggles and lab coat.Schiff’s Reagent: Use extreme caution, Basic fuchsin (pararosaniline) is aknown carcinogen. Wear gloves, goggles, particle mask and lab coat, whilepreparing solution. Work under the hood, keep hot, uncapped, solutionsunder the hood..Avoid contact with and inhalation of dyes.CARBOHYDRATESPAS/D Page: 2 of 2

PROCEDURE:1. Deparaffinize and hydrate to distilled water.2. Warm the diastase solution in the microwave for 20 seconds.3. Place the slide labeled "PAS/D" in the warm diastase solution and intothe waterbath for 15 minutes, do not over digest.4. Rinse in running tap water, rinse in distilled.5. Stain both slides simultaneously, following the PAS procedure.RESULTS:Glycogen will be stained magenta on the PAS stained slide and will beabsent on the PAS/D stained slide.Note: If slide is over digested, the tissue must be recut. Over digestion hasthe appearance of lace; there is no tissue left.NOTES:1. Do not celloidin the slides prior to digestion, it coats the tissue anddigestion can not occur.2. Saliva can be substituted, rinse out mouth, lay slide horizontally instaining dish, in the heat for 30 minutes.3. If slide is over digested, the tissue must be recut. Over digestion hasthe appearance of lace - there is no tissue left.REFERENCES:Bancroft J, Stevens A, Theory and Practice of Histological Techniques, 2ndEd, 1980, pp 187-188, Churchill-Livingstone, NYCarson F, Histotechnology, A Self-Instructional Text,1st Ed, 1990, pp122-123, ASCP, IllCrookham,J, Dapson, R, Hazardous Chemicals in the HistopathologyLaboratory, 2nd ED, 1991, Anatech


PIPARIA

FAT - SUDAN BLACK B-PROPYLENE GLYCOL

PURPOSE: For the demonstration of fat.PRINCIPLE: Sudan Black is slightly basic dye and will combine withacidic groups in compound lipids, thus staining phospholipids also. Analternative stain to the Sudan Black B stain.CONTROL: Use a positive control of a fat smeared slide, and a negativecontrol slide of a paraffin processed tissue, such as lung.FIXATIVE: 10% formalin.TECHNIQUE: Cut frozen tissue sections 10μ.EQUIPMENT: Cryostat, coplin jars. (Making stain, stir plate, filter paper,fritted glass filter, and vacuum) and a 60°C oven. Rinse all glassware in DIwater.REAGENTS:Propylene Glycol:Place in two coplin jars, label #1and #2, can be reused.CAUTION: Avoid contact and inhalation.85% Propylene Glycol:Propylene glycol 85.0 mlDistilled water 15.0 mlCAUTION: Avoid contact and inhalation.Hematoxylin:Commercial Gill-3Glycerin JellySudan Black B/Propylene:Sudan Black B 0.7 gmPropylene glycol 100.0 mlDissolve sudan black in propyleneglycol, slowly, while stirring. Heatto 100°C, but not over 110°C, for afew minutes, stirring constantly.Filter through Whatman #2 filterpaper. Cool, and filter again through

a frittered glass filter of mediumporosity with suction. Store in a60°C oven. Solution stable for 1year.CAUTION: Avoid contact and inhalation.SAFETY: Wear gloves, goggles and lab coat. Avoid contact and inhalation.Propylene glycol; mild skin and eye irritant. Cumbustible.SPECIAL CELLS AND TISSUESSUDAN BLACK B Page: 2 of 2PROCEDURE:1. Pick-up frozen sections on clean glass slides if fresh, albuminizedslides if fixed.2. Fix slides in 10% formalin if fresh.3. Wash well it tap, rinse in distilled, drain off excess water.4. Propylene glycol, two changes, 5 minutes each.5. Sudan Black, 7 minutes, agitate.6. 85% Propylene glycol, 3 minutes.7. Rinse in distilled water.8. Nuclear Fast Red, 3 minutes.9. Wash in water.10. Wash in tap water, rinse in distilled.11. Mount with aqueous mounting media, Glycerin Jelly.RESULTS:Fat blue-blackNuclei redREFERENCE:Carson, F, Histotechnolog: A Self-Instructional Text, 1st Ed,1990, pp161-62, ASCP PressCrookham,J, Dapson,R, Hazardous Chemicals in the HistopathologyLaboratory, 2nd ED, 1991, Anatech


PIPARIA

MAST CELLS - TOLUIDINE BLUE

PURPOSE: These cells are found widely distributed in the connectivetissue. Their cytoplasm contains granules composed of heparin andhistamine. These granules are metachromatic.PRINCIPLE: Mast cells should stain red-purple (metachromatic staining)and the background stain blue (orthochromatic staining). Metachromasia,tissue elements staining a different color from the dye solution, is due tothe pH, dye concentration and temperature of the basic dye. Blue or violetdyes will show a red color shift, and red dyes will show a yellow colorshift with metachromatic tissue elements.CONTROL: SkinFIXATIVE: 10% formalinTECHNIQUE: Cut paraffin sections 4μ.EQUIPMENT: Coplin jars, rinse all glassware in DI water.REAGENTS:Toluidine BlueStock Solution:Toluidine Blue O 1.0 gm70% alcohol 100.0 mlMix, solution is stable for 6 months.CAUTION: Avoid contact and inhalation.Working Solution:Toluidine blue, Stock 5.0 ml1% Sodium chloride 45.0 ml

Make fresh, discard after use.CAUTION: Avoid contact and inhalation.1% Sodium Chloride:Sodium chloride 0.5 gmDistilled water 50.0 mlMake fresh.SPECIAL CELLS AND TISSUESTOLUIDINE BLUE Page 2 of 2SAFETY: Wear gloves, goggles and lab coat. Avoid contact and inhalation.Toluidine Blue O; Gastrointestinal and blood disorders reported in humans.PROCEDURE:1. Deparaffinize and hydrate to distilled water.2. Working Toluidene blue, 1-2 minutes.3. Rinse in distilled water, 3 changes.4. Dehydrate quickly through 95% and absolute alcohols.5. Clear in xylene, coverslip.RESULTS:Mast cells violetBackground shades of blueREFERENCES:Sheehan D, Hrapchak B, Theory and practice of Histotechnology, 2nd Ed,1980, pp282,Battelle Press, OhioLuna L, Manual Of Histologic Staining Methods from the AFIP, 3rd Ed, 1968,pp 162-163, McGraw-Hill, NYCrookham,J, Dapson,R, Hazardous Chemicals in the HistopathologyLaboratory, 2nd ED, 1991, Anatech


PIPARIA

DOWNY: BLOCK SOFTENING SOLUTION

PURPOSE: Used for soaking paraffin blocks that are dry, brittle, and hardto cut.

REAGENT:Downy Fabric Softener 5.0 ml(obtain through Hospital Stores)Distilled water 100.0 mlMix well, place in labeled specimencontainer. Stable for 2 months.

PROCEDURE1. Face into block.2. Place face down into solution.3. Allow to soak for 5-10 minutes.4. Can be used to wipe the face of the block prior to each section.a. Squeeze out all excess solution from Kim Wipe.b. wipe face of block, section, repeat for as many sections as needed onribbon.

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Special Stain Final