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Blocking is essential for preventing non-specific binding of antibodies or other reagents to the tissue. Even if the antibody has high specificity towards the target, intermolecular forces can promote non-specific binding to other molecules. Consequently, non-specific binding prevents visualization of the antigen-antibody binding of interest. To mitigate nonspecific binding, a blocking step should be carried out before incubation with the primary antibody.
Non-specific ionic bindings are due to, for example, Van der Waals interactions, dipole-dipole interactions or net charges of specific amino acid groups. In this case, altering the ionic strength of the antibody dilution buffer can help to reduce unspecific ionic bindings.
When using a horseradish peroxidase (HRP)- or alkaline phosphatase (AP)-conjugated antibody for detection, the endogenous levels of the enzyme have to be blocked. This normally applies to tissues such as kidney, liver, intestine, lymphoid tissue, etc. Peroxidase can be blocked with buffers containing H₂O₂ and AP can be blocked with buffers containing acetic acid or Levamisole.
In general, serum (same species as the secondary antibody) or bovine serum albumin (BSA) is used for blocking. Sera and BSA can help to prevent unspecific binding to the many hydrophobic side chains of proteins present in the tissue. If you are staining with multiple antibodies, you need to use blocking serum against all used secondaries. If BSA is used, the addition of 0.1–0.5% Triton-X or Tween can help to prevent unspecific binding.
Choose the best blocking solution while working with your negative and positive control samples to set up the threshold of background staining.
Use your blocking buffer to prepare your antibody dilution.
Make sure that your blocking buffer will not interfere with your signal detection method.