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The hydrogen peroxide reacts with and blocks endogenous peroxidase enzyme that may exist in your tissue sample.

In the final step you react DAB to a horseradish peroxidase (HRP) that is conjugated to a secondary (or primary) antibody. The HRP binds the DAB and turns it a rust color so that you can see if/where your protein of interest is located. If there is endogenous peroxidase in the sample it will bind and show rust color as well, giving a false positive.

The methanol is just a stabilizer that keeps the H202 from turning into 2H2O and 02. You can also make a hydrogen peroxide and water solution but it needs to be made fresh each day.

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Q: Role of methanol and hydrogen peroxide in IHC?
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