DAB substrate kit ab64238 is used for IHC staining using HRP / peroxidase enzyme for detection. Reagent is stable for up to 6 hours after mixing the two components.

DAB (3,3"Diaminobenzidine) is the most commonly used chromogen for immunohistochemical staining. In the presence of HRP / peroxidase, DAB produces a brown precipitate that is insoluble in alcohol.

This product is a two component form consisting of a liquid, refrigerator stable DAB Chromogen and DAB Substrate.

IHC protocol suitable for use with DAB substrate kit:For frozen sections, skip steps 1 and 2.

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

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

3. Add enoughhydrogen peroxide blocking solutionto cover the sections. Incubate for 10 minutes. Wash 2 times in buffer.If necessary, useavidin biotin blocking.

4. Applyprotein block(or normal serumfrom 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 inantibody diluentand incubate.

6. Wash 4 times in buffer. Incubate slide withbiotinylated 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 ab64238 orAEC substrateand incubate until desired color is achieved (1-10 mins). Rinse 4 times in buffer.

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

10. Rinse 7-8 times in tap water. Addmounting mediumto cover the section.

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