Immunohistochemistry: Reagents

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Research At NIEHS - Intramural Laboratories & Branches Cellular & Molecular Pathology Pathology Support Core Support Immunohistochemistry Equipment Images Protocols Reagents Resources Retrievals All Scientists All Laboratories	Reagents Immunohistochemistry The Immunohistochemistry staff provides the specifications for solution preparation and the contact information for the companies who supply the reagents. 1X Wash Buffer (pH 7.6) 3% Hydrogen Peroxide Antibody Diluent Chromogen Labeling with Diaminobenzidine (DAB) Chromogen Labeling with Aminoethylcarbazole (AEC) 0.1M Citrate Buffer Antigen Retrieval Solution (pH 6.0) Boric Acid-Borate Buffer (pH 7.6) 0.5M TRIS-HCl (pH 7.8 in 1% CaCl2) 2N HCl PLP Fixative 4% Paraformaldehye (PF) Fixativee 2% Paraformaldehye (PF) Fixative 5X EDTA Decolorizing Solution Hematoxylin 1X Wash Buffer (pH 7.6) Dako Wash Buffer 10X is supplied as a 1 L concentrated Tris-buffered saline solution (10X) containing Tween 20, pH 7.6 (±0.1). It is sufficient for preparing 10 L of working solution. Prepare 1X Wash Buffer by mixing the 1-L 10X concentrate with 9 L of distilled water. Supplies Dako Wash Buffer 10X Dako North America, Inc http://www.dakousa.com Carpinteria, CA 93013 Tel (800) 235-5763 Code S3006 Back to top 3% Hydrogen Peroxide Prepare 200 ml 30% Hydrogen Peroxide 20 ml Distilled Water 180 ml Supplies 30% Hydrogen peroxide Fisher Scientific http://www.fisherscientific.com/ Pittsburg, PA Tel (800) 766-7000 Catolog No. H325-500 Back to top Antibody Diluent Prepare 500 ml Bovine serum albumin (BSA) 5 gm 1X Automation buffer 500 ml Mix BSA with 1X automation buffer and refrigerate. Supplies Albumin, bovine (BSA) Jackson ImmunoResearch http://www.jacksonimmuno.com/ West Grove, PA Tel (800) 367-5296 Catalog No. 001-000-162 10X Automation Buffer Biomeda Corporation Order from Fisher Scientific http://www.fisherscientific.com/ Pittsburg, PA Tel (800) 766-7000 Catalog No. BM-M30 Back to top Chromogen Labeling with Diaminobenzidine (DAB) Prepare 1 drop DAB Chromogen per ml Buffered Substrate Procedure Wipe excess buffer from around the tissue section. Apply DAB chromogen and incubate 6 minutes at room temperature in the dark. Discard excess DAB into a waste hazard bottle. Rinse slides in running tap water for 3 min. Counterstain in Harris Hematoxylin for 30 seconds using constant agitation. Filter prior to use. Wash in tap water until water is clear. Place slides in 1X automation buffer until sections are blue (1 min). Rinse in tap water. Dehydrate the tissue sections through 95% and 100% ethanol to xylene. Apply mounting medium (Permount) and coverslip. Results: antigenic sites - brown. Supplies: Dako Liquid DAB Substrate-Chromogen System Dako Corporation http://www.dakocytomation.com/ Carpinteria, CA Tel (800) 235-5763 Catalog No. K3466 Hematoxylin Allegience (VWR) http://www.vwr.com Tel (800) 964-5227 Catalog No. Richard Allen #72711 Permount Fisher Scientific http://www.fisherscientific.com/ Pittsburgh, PA Tel (800) 766-7000 Catalog No. SP15-100 Back to top Chromogen Labeling with Aminoethylcarbazole (AEC) Rinse slides briefly with distilled water and remove excess liquid from the surface of the slide. Apply AEC substrate and incubate 45 min. at room temperature. Rinse slides in distilled water. Counterstain in Harris Hematoxylin for 2 min. Wash in tap water. Place slides in 1 X automation buffer until sections are blue (2 min). Wash in tap water and allow slide to air dry. Apply an aqueous based mounting media (Advantage Permanent Mounting Media) and coverslip. AEC will disolve in organic solvents and thus result in the loss of positive staining; therefore, use an aqueous based mounting media. Results: antigenic sites - pink to red. Supplies AEC Peroxidase Chromagen Kit* Biomeda Corporation http://www.fisherscientific.com/ Foster City, CA Tel (800) 341-8787 Catalog No. S01 Hematoxylin Allegience (VWR) http://www.vwr.com Tel (800) 964-5227 Catalog No. Richard Allen #72711 Aqueous Mounting Media Advantage Permanent Mounting Media INNOVEX Biosciences http://www.innovex.com/ Richmond, CA Tel (800) 622-7808 Catalog No. NB300 Back to top 0.1M Citrate Buffer Antigen Retrieval Solution (pH 6.0) Prepare 1L 10X ANTIGENDECLOAKER 100 ml Distilled Water 900 ml Supplies: ANTIGENDECLOAKER Biocare Medical http://www.biocare.net/ Walnut Creek, CA Tel (800) 799-9499 Catalog No. CB910MM Back to top Boric Acid-Borate Buffer (pH 7.6) Stock Solution A: Boric Acid 1.24 gm/100 ml distilled water Stock Solution B: Sodium Biborate 1.9 gm/100 ml distilled water Solution A ......... 85 ml Solution B ......... 15 ml Mix solutions A and B. Stable at room temperature. Supplies: Boric acid (H3BO3) Mallinckrodt Specialty Chemical Co. http://www.mallinckrodt.com/ Paris, KY Tel (314) 654-2000 Catalog No. 2549 Sodium biborate (Na2B4O7.10H2O) J.T. Baker Inc. http://www.jtbaker.com/ Phillipsburg, NJ Tel (800) 582-2537 Catalog No. 3570-01 Back to top 0.5M TRIS-HCl (pH 7.8 in 1% CaCl2) TRIS 6.057 gm Distilled water 800 ml CaCl2 1.0 gm Mix TRIS, distilled water and CaCl2. pH to 7.8 with 1N HCl. Bring up to volume (1L) with distilled water. This solution is stable at room temperature for one month. Supplies: TRIS, Ultra pure (C4H11NO3) ICN Biochemicals http://www.icnpharm.com/ Aurora, OH Tel (800) 831-3000 Catalog No. 819620 Calcium chloride Dihydrate (CaCl2.2H2O) Mallincrodt Specialty Chemical Co. http://www.mallinckrodt.com/ Paris, KY Tel (314) 654-2000 Catalog No. 4160 Back to top 2N HCl Distilled water 834.0 ml Concentrated HCl 166.0 ml Prepare under a hood. Add HCl slowly to the water. Stable at room temperature. Supplies: Hydrochloric acid (HCl) (37%) Mallincrodt Specialty Chemical Co. http://www.mallinckrodt.com/ Paris, KY Tel (314) 654-2000 Catalog No. H613 Back to top PLP Fixative PLP Fixative Makes approx 800 mL of fixative Part A - Making stock solutions. (All soln. for part A are made the same day as part B, which is one day prior to Part C) 8% Paraformaldehyde Weigh out 16g of Paraformaldehyde Measure out 200 mL of distilled water Add 160mL of the distilled water to the 16g paraformaldehyde Allow mixture to stir for at least 2hr at 37-50° After time has elapsed, add dropwise 10 N NaOH to clear soln. Slowly add remaining 40mL of water. Filter soln. and store in a dark bottle at 4°C 2M lysine-HCL prepared by dissolving 36.5g of lysine-HCl in 1L of distilled water 0.1M anhydrous dibasic sodium phosphate prepared by dissolving 14.2g of Na2HPO4 in 1L of distilled water 0.1M monobasic sodium phosphate (not anhydrous) prepared by dissolving 6.9g of NaH2PO4 in 500mL distilled water Part B - Making the fixative Take 250ml of Lysine-HCl (#2) and use the dibasic soln (#3) to bring the lysine-HCl soln to a pH=7.4. Take 250ml of the dibasic soln (#3) and use the monobasic soln (#4) to bring the dibasic soln to a pH=7.4 Take soln #5 and add enough of soln #6 to bring the volume to 500mL Repeat steps 5, 6, and 7 and combine the solutions for a total of 1000mL Store mixture at 4°C Part C - Day of use. Use 600ml of soln lysine-phosphate soln (#8) and add 200mL of the paraformaldehyde soln (#1); allow to stir Add 1.712g of sodium periodate right before use. (Standard measurement .214g per 100 mL of fixative) Back to top 4% Paraformaldehye (PF) Fixative Always prepare fresh Prepare 100 mls 4% Paraformaldehyde Dissolve 4 g. of paraformaldehyde in 90 ml. of 1X PBS Heat gently to 58-60°C under a hood. Do not heat over 60°C (PF dissociates @ 60°C) Add 10N NaOH to clear the solution (pH 10 dissolves the paraformaldehyde). Usually 5-10 drops Remove from heat and pH Carefully pH to pH 7.0-7.5 Bring to volume (100 mls) PBS (for a final concentration of 4 g. in 100 ml. of 1x PBS) Filter sterilize through a .22 micron filter Use at 4°C for RNA work. Can use at room temperature for non-RNA work Back to top 2% Paraformaldehye (PF) Fixative Always prepare fresh Prepare 100 mls 2% Paraformaldehyde Dissolve 2 g. of paraformaldehyde in 90 ml. of 1X PBS Heat gently to 58-60°C under a hood. Do not heat over 60°C (PF dissociates @ 60°C) Add 10N NaOH to clear the solution (pH 10 dissolves the paraformaldehyde). Usually 5-10 drops Remove from heat and pH Carefully pH to pH 7.0-7.5 Bring to volume (100 mls) PBS (for a final concentration of 4 g. in 100 ml. of 1x PBS) Filter sterilize through a .22 micron filter Back to top 5X EDTA Preparation for use Dilute 1 parts EDTAdecloaker is to 4 parts deionized (DI). The solution is blue at room temperature. When heating, if the solution turns greenish blue, green or yellow, the pH of the solution is not optimum and the tissue staining may be significantly reduced. The pH of the 1X solution should range from 8.0 to 8.5 for optimum staining. The grade of the DI water is very important. If high-grade water is difficult to obtain, sterile water for injection can be purchased from the hospital pharmacy or DI water can be purchased from VWR/Baxter. Make prior to use and do not store ready-to-use. Back to top Decolorizing Solution 200 mL of 70% ethanol 500 µL of HCl (concentrated) Mix ethanol and HCl Submerge slides in solution for 2 minutes Rinse slides with distilled water Rinse slides in 1X automation buffer Back to top Hematoxylin Hematoxylin should be filtered daily to minimize the collection of crystals. Supplies Hematoxylin 7211 Richard-Allan Scientific (Part of Thermo Fisher Scientific) http://www.rallansci.com Kalamazoo, MI 49008 Tel (800) 522-7270 Catalog No. 7211 Back to top
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This page URL: http://www.niehs.nih.gov/research/atniehs/labs/lep/path-support/immuno/reagents.cfm NIEHS website: http://www.niehs.nih.gov/ Email the Web Manager at webmanager@niehs.nih.gov Last Reviewed: September 05, 2008