Precipitable peroxidase substrate for immunoblotting and immunohistochemical staining techniques supplied as a 50X concentrate (0.095 mol/l). To be used with AEC Substrate Buffer, Cat. No. 152226
Precipitable peroxidase substrate for immunoblotting and immunohistochemical staining techniques. Produces a red end product that is soluble in alcohol. When used in immunohistochemistry, aqueous mounting media, and counterstains must be used. Directions included for use in each application.
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Okano, K., et al. 1992. Rinsho Byori 40, 93.
Krenacs, T., et al. 1990. Histochem. J. 22, 530.
Stable at 4°C and 18-26°C. Functionality determined by performing an immunohistologic stain using the final product.
Suggested procedure for use of AEC in immunohistochemistry and immunoblotting:
1. Complete all required incubations with antibodies and HRP-labeled probes, and all necessary wash steps.
2.Prepare 1X AEC Substrate Buffer: Add 9 parts distilled water to 1 part 10X AEC Substrate Buffer, Cat. No. 152226 (0.5 M acetate buffer containing 88 mM H2O2). Mix well. The final pH of this solution will be ~5.0.
3. Prepare 1X AEC Substrate Solution: Add 1 part 50X AEC Concentrate to 50 parts 1X AEC Substrate Buffer (prepared in Step 2). Mix well.
4. Following the final wash, completely cover tissue sections with 1X AEC Substrate Solution (prepared in Step 3) and incubate 10-30 min at room temperature. The same conditions apply for immunoblotting. Note: Variables associated with assay conditions will dictate the proper reaction times.
5. After reaction is complete, wash tissue sections or blotting membranes thoroughly in distilled water.
6. If tissue sections require a counterstain, Mayer′s Hematoxylin followed by blueing in alkaline tap water or a 0.5% aqueous methyl green solution is recommended.
7. Mount tissue sections in a water-based mounting medium. Air-dry blots and store protected from light.