AEC substrate kit for use in IHC using HRP / peroxidase detection. Ready to use formulation.

Previously called AEC Substrate System (Ready to Use).

IHC protocol suitable for use with AEC Substrate Kit ab64252:

1. Deparaffinize and rehydrate formalin-fixed paraffin-embedded tissue section.

2. Use appropriate antigen retrieval buffer or enzyme (primary antibody dependent) to treat sections. Wash 3 times in buffer.

3. Add enough hydrogen peroxide blocking solution to cover the sections. Incubate for 10 minutes. Wash 2 times in buffer. If necessary, use avidin biotin blocking.

4. Apply protein block (or normal serum from same species as secondary antibody) and incubate for 5 minutes at room temperature to block nonspecific background staining. Wash once in buffer.

5. Apply primary antibody in antibody diluent and incubate.

6. Wash 4 times in buffer. Incubate slide with biotinylated secondary antibody (or HRP polymer secondary antibody and skip step 7). Wash 4 times in buffer.

7. Apply streptavidin-HRP and incubate for 10 minutes at room temperature.

8. Rinse 4 times in buffer. Place slide in DAB substrate or AEC Substrate ab64252 and incubate until desired color is achieved (1-10 mins, recommended 10 mins for AEC). Rinse 4 times in buffer.

9. Add enough drops of hematoxylin to cover the section. Incubate for 1 minute.

10. Rinse 7-8 times in tap water. Add mounting medium to cover the section.

Find complete IHC kits, and reagents for antigen retrieval, blocking, signal amplification, visualization, counterstaining, and mounting in the IHC kits and reagents guide.