The Immunohistochemistry staff provides the specifications for solution preparation and the contact information for the companies who supply the reagents.
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
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
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
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
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.