Role of methanol and hydrogen peroxide in IHC?

Answer 1

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.

Answer 2

Methanol is used in immunohistochemistry (IHC) as a dehydration agent to remove water from tissue sections before staining. Hydrogen peroxide is used to block endogenous peroxidase activity, preventing non-specific background staining in IHC. Both reagents are crucial for achieving accurate and reliable results in IHC staining.