Antibody Diluent ab64211 is used to dilute primary antibodies to their working concentrations for use in IHC. It can also be used for convenient storage of diluted primary antibodies.

The antibody diluent is formulated to reduce background and non-specific binding of the primary antibody. In most cases, antibodies can be stored in the diluent, at their working concentrations for IHC, for up to 24 months at 2-8°C.

The diluent is based on a Tris buffer, and contains a stabilizing carrier protein, a wetting agent to reduce surface tension, an anti-microbial agent, and a green dye. It should not be used with HRP-conjugated primary antibodies.

IHC protocol suitable for use with Antibody Diluent ab64211:
For fluorescent IHC, skip step 3, incubate with fluorescent dye conjugated secondary at step 6, skip rest of steps, and mount with anti-fade mounting medium.

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 ab64211 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.