What buffer do you recommend for blocking in an IF experiment?

IF-approved antibodies from CST are validated for use with a blocking buffer that contains 5% serum diluted in PBS containing 0.3% Triton X-100. Serum components offer diversity over alternatives such as milk and BSA and may bind and occupy more sources of non-specific protein-protein interaction. Choosing serum from the same species as the host species of your secondary antibody (e.g., goat, donkey) may also help to reduce background originating from endogenous Fc receptors.
 
At CST, we offer fluorochrome-conjugated F(ab’)2 fragment secondary antibodies that reduce the potential for non-specific binding from this source. Therefore, if you are using one of our fluorochrome-conjugated F(ab’)2 fragment secondary antibodies, blocking may not be necessary.

Last updated: April 29, 2026