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Avidin Biotin Blocking Kit ab64212 blocks signal from endogenous avidin, biotin and biotin-binding proteins in tissues when used with biotin-based IHC detection (eg. with ABC IHC detection kits).

When using the kit, firstly an excess of avidin is added to the sample to bind endogenous biotin, that avidin is then blocked with an excess of biotin. Excess biotin and avidin is washed away.

The kit is often used with cells and tissues containing high levels of biotin. This can be indicated by blocking sections with hydrogen peroxide, and then incubating sections with streptavidin-HRP and then DAB; brown DAB staining indicates endogenous biotin. Kidney, liver, spleen especially contain high levels of biotin.

This kit was previously called Endogenous Avidin/Biotin Blocking Kit.

IHC protocol suitable for use with Avidin Biotin Blocking Kit ab64212:For frozen sections, skip steps 1 and 2.

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, block for endogenous biotin by incubating with avidin block for 15 mins, washing twice, incubating with biotin block for 15 mins, and washing twice.

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 and incubate until desired color is achieved (1-10 mins). 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.