Avidin/Biotin Blocking Kit from Vector Laboratories

Non-specific background is one of the most problematic issues encountered when detecting proteins by immunostaining in tissue sections. More often than not, such unwanted background can mask the real immunosignal, which can make identification and interpretation of specific labeling difficult. There are various methods to reduce background. For example, a hydrogen peroxide step is usually included, which quenches endogenous peroxidase activity and allows for much cleaner detection of immunosignals when using HRP-linked secondary antibodies. However, in some cases, this is not enough and additional suppression of background is needed.

Our lab has been using the Avidin/Biotin Blocking Kit from Vector Labs for our immunohistochemical staining procedures for several years with great success. Our work involves a considerable amount of immunostaining using tissue sections cut from formalin-fixed, paraffin-embedded (FFPE) mouse and human tissue samples. One of our major issues has been non-specific background staining, which we have primarily observed when using mouse monoclonal antibodies on mouse tissue. This issue was partially resolved by using the Mouse-On-Mouse (M.O.M) Peroxidase Kit from Vector Labs. However, we were still seeing background immunostaining in renal tissue, our primary tissue of interest.

We have now overcome this problem with the utilization of a routine avidin/biotin blocking step in our standard immunohistochemistry protocol, which has enabled us to visualize specific immunostaining with very low background. The blocking step is included after the primary antibody step, and prior to the addition of HRP-conjugated secondary antibody, as a 15 minute incubation with avidin followed by a 15 minute incubation with biotin. These avidin/biotin incubations block the endogenous avidin and biotin binding sites in the tissue, which may produce significant background when streptavidin/biotin-based chromogenic substrates are used for visualizing the specific immunosignal. In our hands, this kit works very well and has virtually eliminated background staining caused by avidin/biotin binding site issues.