DEPARTMENT OF PATHOLOGY SBKS 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 in mesotheliomas, certain amounts occur normally in blood vessel walls but increase in early lesions of atherosclerosis. PRINCIPLE: Alcian blue is a group of polyvalent basic dyes that are water soluble. 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 and carboxylated acid mucopolysaccharides and sulfated and carboxylated sialomucins (glycoproteins). It is believed to form salt linkages with the acid groups of acid mucopolysaccharides. CONTROL: Small intestine, appendix, or colon. FIXATIVE: 10% NBF, Bouins, or Hollande's. TECHNIQUE: 4m paraffin sections. EQUIPMENT: Rinse glassware in DI water. Coplin jars, pH meter, REAGENTS: 3% Glacial Acetic Acid Acetic acid 3.0 ml Distilled water 100.0 ml Solution is stable for 1 year.
CAUTION: Contains acid.

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

Nuclear Fast Red (Kernechtrot): Aluminum sulfate 25.0 gm Distilled water 500.0 ml Nuclear fast red 0.5 gm Dissolve the aluminum sulfate in the water. Add the nuclear fast red, dissolve with aid of heat. Filter, add a crystal of thymol. Stable for 1 year.
CAUTION: IRRITANT avoid contact and inhalation

CARBOHYDRATES ALCIAN BLUE PROCEDURE: 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: blue Nuclei (using Nuclear fast red) reddish pink

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE PIPARIA IRON - PRUSSIAN BLUE REACTION - MALLORY'S METHOD
PURPOSE: To demonstrate ferric iron in tissue sections. Small amounts of iron are found normally in spleen and bone marrow. Excessive amounts are present in hemochromatosis, with deposits found in the liver and pancreas, hemosiderosis, with deposits in the liver, spleen, and lymph nodes. PRINCIPLE: The reaction occurs with the treatment of sections in acid solutions of ferrocyanides. Any ferric ion (+3) in the tissue combines with the ferrocyanide and results in the formation of a bright blue pigment called 'Prussian blue" or ferric ferrocyanide. CONTROL: A known positive control tissue. FIXATIVE: 10% formalin TECHNIQUE: Cut paraffin sections 4 . EQUIPMENT: Microwave oven, acidcleaned glassware, non-metalic forceps. REAGENTS: 5% Potassium Ferrocyanide: Potassium ferrocyanide 25.0 gm Distilled water 500.0 ml Mix well, pour into an acid-cleaned brown bottle. Stable for 6 months.
CAUTION: Low toxicity if not heated..

5% Hydrochloric Acid: Hydrochloric acid, conc. 25.0 ml Distilled water 475.0 ml Mix well, pour into brown bottle, stable for 6 months.
CAUTION: Corrosive, avoid contact and inhalation.

Working Solution: 5% potassium ferrocyanide 25.0 ml 5% hydrochloric acid 25.0 ml Make fresh, discard after use. . PROCEDURE: 1. Deparaffinize and hydrate to distilled water. 2. *Working solution, * microwave, 30 seconds. Allow slides to stand in solution 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) blue Nuclei red Background pink

Nuclear-fast Red:

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE PIPARIA

FITE'S ACID FAST STAIN - LEPROSY

PURPOSE: To demonstrate mycobacterium leprae (leprosy), which are acid fast organisms. PRINCIPLE: This technique combines peanut oil with the deparaffinizing solvent (xylene), minimizing the exposure of the bacteria's cell wall to organic solvents, thus protecting the precarious acid-fastness of the organism. CONTROL: Leprosy positive tissue. FIXATIVE: 10% formalin TECHNIQUE: Cut paraffin sections 45 microns. EQUIPMENT: Rinse all glassware in DI water, coplin jars, bibulous blotting paper. REAGENTS: Xylene/Peanut Oil Solution: Xylene 50.0 ml Peanut Oil 50.0 ml Mix well. Label with date and initials, solution is stable for 1 year.
CAUTION: Flammable, irritant.

Target organ effects via inhalation on skin, respiratory, reproductive and fetal systems. Methylene blue: produced deleterious effects on fertility in rats. PROCEDURE: 1. Deparaffinize in xylene/peanut oil mixture, 2 changes, 10 minutes each. 2. Drain slides, blot off excess oil. 3. Rinse in distilled water until slide clears. 4. Carbol-fuchsin, 30 minutes, room temperature. 5. Wash in tap water. 6. Acid alcohol until pale pink, dip until stain stops running. 7. Wash in tap water. 8. Counterstain in Working Methylene blue, 30 seconds. 9. Wash in tap water. 10. Blot and air dry. 11. Dip in xylene and coverslip. RESULTS: Acid-fast bacilli red Background blue NOTE: Mineral oil may be substituted for peanut oil. PROCEDURE CARD

Ziehl-Neelsen Carbol-Fuchsin: 1% Acid Alcohol: Working Methylene Blue: FITE'S Page: 2 of 2 Hydrochloric acid: strong irritant to skin, eyes and respiratory system.

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE PIPARIA

RETIC - GORDON AND SWEET'S METHOD - RETICULAR FIBERS
PURPOSE: A silver impregnation technique that demonstrates reticular fibers. Reticulum is a support function of the body and is abundant in liver, spleen, and kidney. In a normal liver the fibers are will defined strands, but necrotic and cirrhotic liver show discontinuous patterns. Reticulum also forms characteristic patterns in relationship to certain tumor cells. PRINCIPLE: The tissue is oxidized, then sensitized with the iron alum, which is replaced with silver. The silver is reduced with formalin to its visible 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 ml Sulfuric acid 3.0 ml Mix well, label with initial and date. Solution is stable for 1 year. Potassium Permanganate: Potassium permanganate 0.5 gm Distilled water 47.5 ml 3% Sulfuric acid 2.5 ml Make fresh, discard after use. 1% Oxalic Acid: Oxalic acid 1.0 gm Distilled water 100.0 ml Mix well, label with initial and date. Solution is stable for 1 year. 2% Iron Alum Ferric ammonium sulfate 2.0 gm Distilled water 100.0 ml Mix well, label with date and initial. Solution is stable for 6 months. 10% Silver Nitrate Stock Silver nitrate 10.0 gm Distilled water 100.0 ml Acid clean all glassware and rinse well. Store in a brown bottle, keep in the refrigerator. Label with initial and date. Stable f or 6 months. 3% Sodium Hydroxide Stock: Sodium hydroxide 1.5 gm Distilled water 50.0 ml Mix well, label with date and initials. Store in the refrigerator, stable for 3 months. Ammoniacal Silver Working Solution: 10% silver nitrate 5.0 ml Add ammonium hydroxide drop by drop until clear again. 3% sodium hydroxide 5.0 ml Add ammonium hydroxide drop by drop until clear again. Distilled water 40.0 ml Using acid clean glassware, agitate solution continually while adding ammonium hydroxide. Make right before use, discard. 10% Formaldehyde: Formaldehyde 5.0 ml Distilled water 45.0 ml Make fresh, discard after use. 0.5% Gold Chloride: Gold chloride 1.0 gm

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE PIPARIA

Distilled water 200.0 ml Use acid clean glassware, label with date and initials. Store in the refrigerator. 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 black Nuclei red 5. Change distilled water after every

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE PIPARIA

LIPOFUSCIN - SUDAN BLACK B

PURPOSE: Lipofuscins, the "wearand-tear" pigment, collects in the more permanent cells (heart, liver, and neurons) of older persons. Lipofuscin is the accumulation of lysosomes, which have absorbed the worn-out, undigestible parts of the cell and known as residual bodies. It is a yellowbrown pigment, and will stain after being processed in paraffin. PRINCIPLE: The lipofuscins react with oil-soluble dyes. CONTROL: Bielschowsky control tissue, cut at 5 . FIXATIVE: 10% formalin TECHNIQUE: Cut paraffin sections 5 . REAGENTS: Sudan Black/Lipofuscin: Sudan Black B saturated 70% alcohol 50.0 ml LIPOFUSCIN - SUDAN BLACK B RESULTS: Lipofuscins, RBC's: black Mix well, filter through filter paper, then filter again through a frittered glass filter of medium porosity with suction. Stable for 6 months. 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

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE 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 fixed to a slide and stained first with Hematoxylin, whi h stains the nuclei c followed by OG-6 and EA-50 or EA-65 as a counterstain. cell 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 spread directly on a clean microscope slide. Sediment (obtained by centrifugation or filtration) from bodily secretions is spread on a clean microscope slide. The smear is immediately fixed with a cyt logical spray fixative or in an o alcohol-ether dip. Fixation or preservation is one of the most important steps in the procedure. Drying of the cells prior to fixation will usually result in artifacts such as nuclear distortion and vacuolization. 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 one hour. A second clean glass slide may be placed on each fixed slide for protection.

Reagents:
EA-50 Multiple Polychrome Stain 1000mL Catalogue # IMEA50 Light Green S.F. Yellowish, Fast Green FCF, Bismark Brown Y, Eosin Y, Phosphotungstic Acid, Glacial Acetic Acid. Filter before use. EA-65 Multiple Polychrome Stain 1000mL Catalogue # IMEA65 Light Green S.F. Yellowish, Fast Green FCF, Bismark Brown Eosin Y, Phosphotungstic Acid, Glacial Acetic Acid. Filter before use. OG-6 Orange G Stain 1000mL Catalogue # IMOG6 Orange G, Phosphotungstic Acid. Filter before use. Harris Hematoxylin 1000mL Catalogue # IMHARRISHX Hematoxylin, 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, a modification of EA-50, has been especially developed for use with OG6 in the study of smears from urinary, paracentetic, thoracentetic material, from sputum, from gastric and external 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 and technique remain the same.

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 water until 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 a salmon 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% ethanol until 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 3 Mounting Procedure: 1. After the smear has been completely cleaned in xylene it is mounted with a microscope 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 air spaces when xylene evaporates. 4. Place the required amount of mounting medium along an edge of one of the longer borders of the coverslip. 5. Place the slide at right angles to the edge of the coverslip so that the side containing the cells is facing the mounting medium.

Storage and Stability:

Store at 15-30C. Reagents are stable until expiration dates shown on the label.

Warnings and Precautions:

Papanicolaou Staining reagents are flammable and toxic. Keep away from sources of ignition. In case of contact with eyes, rinse immediately with water and seek medical advice. May be fatal or cause blindness if swallowed. Dispose of waste in accordance with applicable laws.

Materials Required But Not Provided:

Ethanol Coverslips Hydrochloric Acid Microscope Ammonium Hydroxide Microscope slides Xylene Methanol Page 4

Specimen Collection:
In the collection and preparation of smears for cytological examination, the major objectives are: 1. Specimens should have a sufficient number of cells from the area in question. 2. Smears should contain well preserved cells uniformlydistributed so that each

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE PIPARIA

6. Slowly lower the slide and permit the mounting medium to spread between the slide and coverslip. Cambridge, 1954. 2. United States, Naval Medical School, National Naval Medical Center, Bethesda, Maryland, Manual of gynecological exfoliative cytology. US Government Printing Office, Washington, D.C., 1965. 3. A Manual for Cytotechnology, C. Keebler, J. Reagen, Editors, American Society of Clinical Pathologists Press, Chicago (IL), 1983.

Results:
Nuclei are stained blue while cytoplasm displays varying shades of pink, orange, yellow and green.

Limitations:
1. Proper specimen collection and fixation of cells is essential for interpretation.

References:
1. Papanicolaou GN: Atlas of Exfoliative Cytolo Harvard University gy, Press,

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE 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% ethanol 1 x 3 95% ethanol 1 x 3 80% ethanol 1 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 Hematoxalin Rinse deionized water 1 x 5 Tap water (to allow stain to develop) Dip 8-12x (fast) Acid ethanol (to destain) Rinse 2 x 1 Tap water Rinse 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 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 leave no bubbles. 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. Reagents for H&E staining: Xylene: StatLab (Lewisville, TX) #8400 Laboratory grade, Anapath brand Acid Ethanol: 1 ml concentrated HCl + 400 ml 70% ethanol Hematoxylin: Poly Scientific (Bayshore, NY) #s212A Harris hematoxylin with glacial acetic acid Eosin: Poly Scientific (Bayshore, NY) #s176 Eosin Phloxine stain, working Permount: Fisher Scientific #SP15100 Histological mounting medium

3 x 5 95% ethanol 3 x 5 100% ethanol (blot excess ethanol

before going into xylene)

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE 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 ), and Aniline 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% ethanol 1 x 3 95% ethanol 1 x 3 80% ethanol 1 x 5 deionized H 2O 2. Mordant in Bouin's Solution at room temperature overnight in a hood. Be careful, Bouin's solution is hazardous and the picric acid, when in less than 10% water, is very explosive. 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 in distilled 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) and

Solution B (1.2% Ferric Chloride and 1% Acetic Acid in distilled water). The working solution is good for approximately 10 days. ** Hematoxylin stains nuclei blueblack. 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 been overused 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 1 volume of Phosphotungstic Acid Solution and 1 volume of Phosphomolybdic Acid Solution with 2 volumes of distilled water. Discard after one use. Formation of a precipitate in Phosphomolybdic 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. **Aniline blue stains collagen bl ue. 10. Rinse slides briefly in distilled water. 11. Place slides in 1% acetic acid solution for 3-5 minutes. Discard this solution.

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE PIPARIA

MASSON TRICHROME STAINING

** Rinsing in acetic acid after staining renders the shades of color more delicate and transparent. ** If blue staining of connective tissue appears faded, the section has probably been overdifferentiated in the acetic acid solution. 12. Dehydrate to xylene. 2 x 3 95% ethanol 2 x 3 100% ethanol (blot excess ethanol
before going into 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.

3 x 5 Xylene

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE 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 45 microns thick. EQUIPMENT: Rinse glassware in DI water. Coplin jar, microwave oven. REAGENTS: Alcian Blue, pH2.5: See Alcian Blue 0.5% Periodic Acid: See PAS Schiff's Reagent: See PAS Hematoxylin Gill-3: Purchased through Baxter SAFETY: Wear gloves, goggles, particle mask and lab coat, avoid contact and inhalation. Work under the hood, keep hot, uncapped, solutions under the hood. Schiffs Reagent: Use extreme caution, Basic fuchsin (pararosaniline) is a known 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. CARBOHYDRATES ALCIAN BLUE-PAS Page: 2 of 2 8. 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 blue Neutral polysaccharides magenta REFERENCES: Sheehan D, Hrapchak B, Theory and practice of Histotechnology, 2nd Ed, 1980, pp 163,173-174, Battelle Press, Ohio Bancroft J, Stevens A, Theory and Practice of Histological Techniques, 2nd Ed, 1982,pp194-98,Churchill Livingstone, NY. Carson F, Histotechnology A Self Instructional Text, 1st Ed, 1990,pp130 131, ASCP, Ill Crookham, J, Dapson, R, Hazardous Chemicals in the Histopathology Laboratory, 2nd ED, 1991, Anatech LTD

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE PIPARIA

PAS - McMANNUS' PERIODIC ACID SCHIFF'S GLYCOGEN

PURPOSE: Glycogen is present in skin, liver, parathyroid glands and skeletal and cardiac muscle. The PAS stain is used for demonstration of basement membranes, fungus secreting adenocarcinoma from undifferentiated squamous cell carcinoma, and mucosubstances secreted from the epithelia of various organs. A routine stain for liver and kidney biopsies. PRINCIPLE: The PAS stain is a histochemical reaction in that the periodic acid oxidizes the carbon to carbon bond forming aldehydes which react to the fuchsin-sulfurous acid which form the magenta color. CONTROL: For staining fungus; use a known positive such as those used for the GMS. Use skin, aorta or normal liver for positive PAS staining. FIXATIVE: Any well fixed tissue. TECHNIQUE: Cut paraffin sections 45 m( 3m for kidney biopsies). EQUIPMENT: Rinse glassware in DI water. Coplin jar, microwave oven. REAGENTS: 0.5% Periodic Acid: Periodic acid 0.5 gm Distilled water 100.0 ml Mix well, label with date and initial. Stable for 1 year.
Caution: Avoid contact and inhalation.

Hydrochloric acid 10.0 ml Stir solution for 2 hours, set in a dark cool place overnight. The solution is now a clear light brown to yellow color. Add: Activated charcoal 500.0 gm (or two heaping spoons) Stir. Filter through Whatman #2 filter paper into a 1000 ml graduated cylinder. Change the filter paper often. Restore volume to 1000 ml with distilled water. Store in Refrigerator, solution is stable for 6 months.
CAUTION: Carcinogen, corrosive.

Hematoxylin, GILL-3 Purchased through Baxter. CARBOHYDRATES PAS STAIN Page: 2 of 3 Lillie's Cold Schiff's Reagent: Basic fuchsin 10.0 gm Sodium metabisulfite 18.0 gm Distilled water 1000 0 ml

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 and fetal systems. Corrosive. Schiffs Reagent: Use extreme caution, Basic fuchsin (pararosaniline) is a known carcinogen. Wear gloves, goggles, particle mask and lab coat, while preparing solution. Work under the hood, keep hot, uncapped, solutions under the hood. CARBOHYDRATES PAS STAIN Page: 3 of 3 PROCEDURE: 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, until deep magenta.

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE 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 30 minutes. RESULTS: Glycogen, fungus: magenta Nuclei blue NOTES: 1. To check stain, pour 10 ml of formaldehyde in cylinder, add a few drops of Schiff's, it should turn redpurple 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 redissolved if 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, Ohio Bancroft J, Stevens A, Theory and Practice of Histological Techniques, 2nd Ed, 1982, pp 188-190, Churchill Livingstone, NY Crookham, J, Dapson, R, Hazardous Chemicals in the Histopathology Laboratory, 2nd ED,1991, Anatech

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE PIPARIA PAS/D - GLYCOGEN DIGESTION - DIASTASE
PURPOSE: To determine glycogen by digesting out and staining with PAS stain. PRINCIPLE: The diastase (or a-amylase) act on glycogen to de polymerize it into smaller sugar units, maltose and glucose, that are washed out of the section. CONTROL: Identical sections are obtained on two separate slides. One is digested the other is not, both are stained with the PAS stain. Liver is a good control. FIXATIVE: Any well fixed tissue. TECHNIQUE: Cut paraffin sections at 4m to 5m EQUIPMENT: Rinse glassware in DI water. Coplin jars, 37C oven. REAGENTS: Digestion Solution: Diastase (with a-amylase)0.05 gm (obtained through Ameri. Reagent) Distilled water 50.0 ml Make fresh, use within 15 minutes, discard. 0.5% Periodic Acid: See PAS Schiff's Reagent: See PAS Hematoxylin, Gill-3 Purchased through Baxter SAFETY: Hydrochloric acid is caustic use caution, flush with water. Wear gloves, goggles and lab coat. Schiffs Reagent: Use extreme caution, Basic fuchsin (pararosaniline) is a known carcinogen. Wear gloves, goggles, particle mask and lab coat, while preparing solution. Work under the hood, keep hot, uncapped, solutions under the hood.. Avoid contact with and inhalation of dyes. CARBOHYDRATES PAS/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 into the 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 be absent on the PAS/D stained slide. Note: If slide is over digested, the tissue must be recut. Over digestion has the appearance of lace; there is no tissue left. NOTES: 1. Do not celloidin the slides prior to digestion, it coats the tissue and digestion can not occur. 2. Saliva can be substituted, rinse out mouth, lay slide horizontally in staining dish, in the heat for 30 minutes. 3. If slide is over digested, the tissue must be recut. Over digestion has the appearance of lace - there is no tissue left. REFERENCES: Bancroft J, Stevens A, Theory and Practice of Histological Techniques, 2nd Ed, 1980, pp 187-188, ChurchillLivingstone, NY Carson F, Histotechnology, A SelfInstructional Text,1st Ed, 1990, pp 122-123, ASCP, Ill Crookham,J, Dapson, R, Hazardous Chemicals in the Histopathology Laboratory, 2nd ED, 1991, Anatech

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE PIPARIA FAT - SUDAN BLACK B-PROPYLENE GLYCOL
PURPOSE: For the demonstration of fat. PRINCIPLE: Sudan Black is slightly basic dye and will combine with acidic groups in compound lipids, thus staining phospholipids also. An alternative stain to the Sudan Black B stain. CONTROL: Use a positive control of a fat smeared slide, and a negative control slide of a paraffin processed tissue, such as lung. FIXATIVE: 10% formalin. TECHNIQUE: Cut frozen tissue sections 10 . EQUIPMENT: Cryostat, coplin jars. (Making stain, stir plate, filter paper, fritted glass filter, and vacuum) and a 60C oven. Rinse all glassware in DI water. REAGENTS: Propylene Glycol: Place in two coplin jars, label #1 and #2, can be reused.
CAUTION: Avoid contact and inhalation.

a frittered glass filter of medium porosity with suction. Store in a 60C oven. Solution stable for 1 year.
CAUTION: Avoid contact and inhalation.

85% Propylene Glycol: Propylene glycol 85.0 ml Distilled water 15.0 ml

CAUTION: Avoid contact and inhalation.

Hematoxylin: Commercial Gill-3 Glycerin Jelly Sudan Black B/Propylene: Sudan Black B 0.7 gm Propylene glycol 100.0 ml Dissolve sudan black in propylene glycol, slowly, while stirring. Heat to 100C, but not over 110C, for a few minutes, stirring constantly. Filter through Whatman #2 filter paper. Cool, and filter again through

SAFETY: Wear gloves, goggles and lab coat. Avoid contact and inhalation. Propylene glycol; mild skin and eye irritant. Cumbustible. SPECIAL CELLS AND TISSUES SUDAN BLACK B Page: 2 of 2 PROCEDURE: 1. Pick-up frozen sections on clean glass slides if fresh, albuminized slides 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-black Nuclei red REFERENCE: Carson, F, Histotechnolog: A Self Instructional Text, 1st Ed,1990, pp161 62, ASCP Press Crookham,J, Dapson,R, Hazardous Chemicals in the Histopathology Laboratory, 2nd ED, 1991, Anatech

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE PIPARIA MAST CELLS - TOLUIDINE BLUE
PURPOSE: These cells are found widely distributed in the connective tissue. Their cytoplasm contains granules composed of heparin and histamine. 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 to the pH, dye concentration and temperature of the basic dye. Blue or violet dyes will show a red color shift, and red dyes will show a yellow color shift with metachromatic tissue elements. CONTROL: Skin FIXATIVE: 10% formalin TECHNIQUE: Cut paraffin sections 4 . EQUIPMENT: Coplin jars, rinse all glassware in DI water. REAGENTS: Toluidine Blue Stock Solution: Toluidine Blue O 1.0 gm 70% alcohol 100.0 ml Mix, solution is stable for 6 months.
CAUTION: Avoid contact and inhalation.

Make fresh, discard after use.

CAUTION: Avoid contact and inhalation.

Working Solution: Toluidine blue, Stock 5.0 ml 1% Sodium chloride 45.0 ml

1% Sodium Chloride: Sodium chloride 0.5 gm Distilled water 50.0 ml Make fresh. SPECIAL CELLS AND TISSUES TOLUIDINE BLUE Page 2 of 2 SAFETY: 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 violet Background shades of blue REFERENCES: Sheehan D, Hrapchak B, Theory and practice of Histotechnology, 2nd Ed, 1980, pp282,Battelle Press, Ohio Luna L, Manual Of Histologic Staining Methods from the AFIP, 3rd Ed, 1968, pp 162-163, McGraw-Hill, NY Crookham,J, Dapson,R, Hazardous Chemicals in the Histopathology Laboratory, 2nd ED, 1991, Anatech

DEPARTMENT OF PATHOLOGY SBKS MEDICAL INSTITUTE& RESEARCH CENTRE PIPARIA

DOWNY: BLOCK SOFTENING SOLUTION

PURPOSE: Used for soaking paraffin blocks that are dry, brittle, and hard to cut. REAGENT: Downy Fabric Softener 5.0 ml (obtain through Hospital Stores) Distilled water 100.0 ml Mix well, place in labeled specimen container. Stable for 2 months. PROCEDURE 1. 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 e ach section. a. Squeeze out all excess solution from Kim Wipe. b. wipe face of block, section, repeat for as many sections as needed on ribbon.