For most immunoassays like IHC, ELISA or Western blot, blocking is a crucial step to perform. This procedure blocks spaces that are not occupied by immobilized proteins, which prevents non-specific binding of the antibodies used in the assay. If blocking is not performed, false signals or high background will occur.
There is no universal blocking agent, therefore empirical testing is necessary to determine which agent to use for a given assay.
If normal serum is used as a blocker, it is recommended to use serum derived from the same host species as the detection antibody, conjugated with the reporter enzyme like HRP (Horse radish peroxidase) or ALP (Alkaline phosphatase).
Primary antibody: Rabbit anti-target
Blocking serum: Normal Chicken Serum (usually used at 5 % v/v in PBS with Tween®20 detergent)
Secondary antibody: Chicken anti-Rabbit IgG-HRP conjugated
Note: Rabbit serum cannot be used for blocking in such combination.