You probably know that a highly specific, high affinity primary antibody is key to a successful IHC. But did you know that the companion reagents such as buffers, which establish the pH and ionic strength of the system, are just as important? These reagents can influence the binding of the primary antibody to its epitope and dramatically affect the outcome of the assay.
To help you pick the best reagents for your assay and make sure those Friday nights in the lab are worth the effort, we will spend the next several videos reviewing how companion reagents affect IHC, and, as an example, we will describe our experience optimizing the protocol for one of our antibodies.
Step One. Antigen Retrieval.
Several methods exist for revealing epitopes that have been massed by cross-linking fixatives like formalin. These include Proteolytic-Induced Antigen Retrieval, which relies on an enzyme like Proteinase K, or Heat-Induced Epitope Retrieval, which uses heat to break apart cross-linked bonds and unwind proteins.