How to reduce the background staining in immunohistochemistry?

To minimize unwanted background staining, a protein blocking step is essential. This step helps prevent nonspecific binding of antibodies, particularly to the Fc portion of primary or secondary antibodies. A recommended agent for protein blocking is 5%–10% normal serum from the same species as the secondary antibody. Alternatively, protein buffers such as 0.1%–0.5% bovine serum albumin, nonfat dry milk or gelatin can also be used. Commercial mixes of synthetic peptides are becoming increasingly popular for this purpose. One can deactivate natural peroxidase enzymes by treating with a solution containing 3% hydrogen peroxide in either methanol or water or use a commercial kit specifically designed for this purpose. Naturally occurring lectins can be neutralized by introducing 0.2 M alpha-methyl mannoside in a diluted buffer solution. Another option is to substitute avidin with either streptavidin. One can also decrease the concentration of the primary antibody utilized during the staining process.