SURGICAL PATHOLOGY - HISTOLOGY Date: STAINING MANUAL - CARBOHYDRATES Page: 1 of 3 CONGO RED - PUTCHLER'S MODIFICATION - AMYLOID PURPOSE: To demonstrate amyloid deposits in tissue sections. PRINCIPLE: Amyloid is homogeneous and eosinophilic, the deposits are extracellular and may become sufficiently large enough to cause damage to surrounding tissues. when stained with the Congo Red Stain the amyloid, with the aide of polarizing lenses, will birefringe an apple green color. under the microscope. CONTROL: Known amyloidosis containing tissue. FIXATION: Carnoy's and absolute alcohol are recommended, 10% NBF or Bouin's. TECHNIQUE: Cut paraffin sections at 10 microns. EQUIPMENT: Rinse glassware in DI water. Coplin jars, funnel and glass wool, graduated cylinder, 1mm pipette, and microwave. REAGENTS: Mayer's Hematoxylin: 1% Sodium Hydroxide Hematoxylin 1.0 gm Sodium hydroxide 1.0 gm Distilled water 1000.0 ml Distilled water 100.0 ml Sodium iodate 0.2 gm Mix well. Label, date and initial. Ammonium or Potassium Store in the refrigerator, stable for aluminum sulfate 50.0 gm 3 months. Discard if a precipitate Citric acid 1.0 gm forms. Chloral hydrate 50.0 gm CAUTION: Corrosive, irritant. Dissolve the hematoxylin in water, heat to boiling, boil for 5 minutes. Remove from heat, add sodium iodate, wait 10 minutes to ripen. Add the remainder of the chemicals in the order given, dissolving each one before adding the next. Label, initial, stable for 1 year. Caution: Avoid contact and inhalation. CARBOHYDRATES CONGO RED Page: 2 of 3 Sodium Chloride, Congo Red Stock Solution, Saturated: Stock Solution, Saturated: Sodium chloride 30.0 gm Congo red stain 1.0 gm Distilled water 200.0 ml Stock sodium chloride 500.0 ml Mix well, then add: Mix well. Date and initial. Solution is stable for 6 months. 100% alcohol 800.0 ml CAUTION: Flammable, avoid contact Label, date and initial. Solution is and inhalation. stable for 1 year. Working Solution: CAUTION: Flammable. Stock Congo red solution 50.0 ml Working Solution: 1% sodium hydroxide 0.5 ml Stock sodium chloride 50.0 ml Filter through glass wool, solution 1% sodium hydroxide 0.5 ml should be a clear, not cloudy Make immediately before use, orange/red. Use immediately, discard. discard after use. SAFETY: Wear gloves, goggles and lab coat. Work in well ventilated area, wear particle mask when preparing dye solutions. Sodium Hydroxide; severe irritant to skin and eyes. Corrosive to skin and eyes. Can cause burns. Congo Red: Developmental abnormalities were produced in mouse fetuses after chronic feeding studies. Causes vascular changes in humans after ingestion. PROCEDURE: 1. Deparaffinize and hydrate to water. 2. Mayer's Hematoxylin for 10 minutes. 3. Wash in tap water until blue. 4. *Working sodium chloride solution, microwave, 20 power, 45 seconds. Allow slides to sit in solution for 2-5 minutes. 5 Place directly into Working Congo red solution, microwave, 20 power, for 45 seconds. Allow slides to sit in solution for 2-5 minutes. 6. Dehydrate rapidly in absolute alcohol, 10 dips, 3 changes. 7. Clear in xylene, coverslip with Permount. *Conventional Method: 4. Working sodium chloride solution, room temperature, 20 minutes. 5. Place directly into Working Congo red solution for 1 hour. CARBOHYDRATES CONGO RED Page: 3 of 3 RESULTS: Amyloid red to pink Nuclei blue NOTES: 1. Sections must be cut at 8 to 10 microns for birefringence. 2. Solution must be filtered through glass wool, not paper filters for birefringence to occur. 3. Tissue fixed in solutions other than formalin may display false positive birefringence. REFERENCES: Stochem JH, Cytochem, 10:355-364 Sheehan D,Hrapchak B, Theory and practice of Histotechnology, 2nd Ed, 1980, pp 175-178, Battelle Press, Ohio Crookham, J, Dapson, R, Hazardous Chemicals in the Histopathology Laboratory, 2nd ED, 1991, ANATECH LTD Prepared: By: Approved: By: Downloaded from WebPath: Internet Pathology Laboratory http://www-medlib.med.utah.edu/WebPath/webpath.html PROCEDURE CARD AMYLOID - CONGO RED - PUTCHLER'S MODIFICATION CONTROL: Known amyloidosis containing tissue. TECHNIQUE: Cut paraffin sections at 10 microns. PROCEDURE: 1. Deparaffinize and hydrate to water. 6. Dehydrate rapidly in absolute alcohol, 2. Mayer's Hematoxylin for 10 minutes. 10 dips, 3 changes. 3. Wash in tap water until blue. 7. Clear in xylene, coverslip with 4. *Working sodium chloride solution, Permount. microwave, 20 power, 45 seconds. * Conventional Method: Allow slides to sit in solution for 2-5 4. Working sodium chloride solution, minutes. room temperature, 20 minutes. 5. *Place directly into Working Congo red 5. Place directly into Working Congo solution, microwave, 20 power, for 45 red solution for 1 hour. seconds. Allow slides to sit in solution for 2-5 minutes. RESULTS: Amyloid red to pink Nuclei blue Mayer's Hematoxylin: Sodium Chloride Hematoxylin 1.0 gm Stock Solution, Saturate: Distilled water 1000.0 ml Sodium chloride 30.0 gm Sodium iodate 0.2 gm Distilled water 200.0 ml Ammonium or Potassium aluminum Mix well, then add: sulfate 50.0 gm 100% alcohol 800.0 ml Citric acid 1.0 gm Label, date and initial. Solution is stable for 1 Chloral hydrate 50.0 gm year. Dissolve the hematoxylin in water, heat to CAUTION: Flammable. boiling, boil for 5 minutes. Remove from heat, add sodium iodate, wait 10 minutes to Working Solution: ripen. Add the remainder of the chemicals in Stock sodium chloride 50.0 ml the order given, dissolving each one before 1% sodium hydroxide 0.5 ml adding the next. Label, initial, stable for 1 Make immediately before use, discard. year. Congo Red 1% Sodium Hydroxide Stock Solution,Saturated: Sodium hydroxide 1.0 gm Congo red stain 1.0 gm Distilled water 100.0 ml Stock sodium chloride 500.0 ml Mix well. Label, date and initial. Store in the Mix well. Date and initial. Solution is stable refrigerator, stable for 3 months. Discard if a for 6 months. precipitate forms. CAUTION: Flammable CAUTION: Corrosive. Working Solution: Stock Congo red solution 50.0 ml 1% sodium hydroxide 0.5 ml Filter through glass wool, solution should be a clear, not cloudy orange/red. Use immediately, discard after use. CONGO RED SODIUM CHLORIDE STOCK SOLUTION, SATURATED: STOCK SOLUTION, SATURATED: Congo red stain 1.0 gm Sodium chloride 30.0 gm Stock sodium chloride 500.0 ml Distilled water 200.0 ml Mix well. Date and initial. Solution Mix well, then add: is stable for 6 months. 100% alcohol 800.0 ml CAUTION: Flammable, avoid contact and inhalation. Label, date and initial. Solution is stable for 1 year. WORKING SOLUTION: CAUTION: Flammable. Stock Congo red solution 50.0 ml 1% sodium hydroxide 0.5 ml WORKING SOLUTION: Filter through glass wool, solution Stock sodium chloride 50.0 ml should be a clear, not cloudy 1% sodium hydroxide 0.5 ml orange/red. Use immediately, Make immediately before use, discard after use. discard. DATE: DATE: TECH: TECH: EXPIRATION: EXPIRATION: MAYER’S HEMATOXYLIN: 1% SODIUM HYDROXIDE Hematoxylin 1.0 gm Sodium hydroxide 1.0 gm Distilled water 1000.0 ml Distilled water 100.0 ml Sodium iodate 0.2 gm Mix well. Label, date and initial. Ammonium or Potassium Store in the refrigerator, stable for aluminum sulfate 50.0 gm 3 months. Discard if a precipitate Citric acid 1.0 gm forms. Chloral hydrate 50.0 gm CAUTION: Corrosive. Dissolve the hematoxylin in water, heat to boiling, boil for 5 minutes. SAFETY/PPE: Wear gloves, goggles Remove from heat, add sodium and lab coat. iodate, wait 10 minutes to ripen. Add the remainder of the chemicals DATE: in the order given, dissolving each one before adding the next. Label, TECH: initial, stable for 1 year. EXPIRATION: SAFETY/PPE: Wear gloves, goggles and lab coat. Work in well ventilated area or under hood. Caution: avoid contact and inhalation. DATE: TECH: EXPIRATION: .