Blocking ICC/IF

What is the purpose of blocking step in ICC/IF?

In ICC/IF, antigen detection is dependent on specific binding of the antibody to its epitope. This binding, in turn, is governed by hydrophobic interactions, ionic interactions, hydrogen bonding, and other intermolecular forces. Besides facilitating specific antigen-antibody binding, the same attractive forces/bonds can also contribute to non-specific binding. The blocking step minimizes non-specific interactions that result in the background and false-positive staining (artifacts). It is important to use reagents and blocking conditions that reduce nonspecific binding while having a negligible impact on specific antigen-antibody binding.

What are the Most Critical Considerations for Blocking Buffers?

Blocking buffer should contain heat-inactivated normal serum from the same species as the host of the secondary antibody. Other, less preferred, blocking agents include fetal calf serum (FCS), bovine serum albumin (BSA, Catalog # 5217), casein protein, non-fat dry milk, and gelatin.
To facilitate entry into the cell and to minimize the effects of non-specific hydrophobic interactions, it is essential to add a non-ionic detergent (Triton X-100 or Tween 20) to the blocking buffer, antibody diluent, and wash buffers. Blocking buffers commonly contain 1X PBS with 0.1% Triton X-100 (PBS-T).
To reduce non-specific ionic interactions, we recommend increasing the ionic strength of the fixative and/or the antibody diluent. However, this is not recommended for buffers containing monoclonal antibodies, which recognize a single epitope, because it may negatively impact binding affinity

Composition of Blocking Buffers

Blocking Agent	User Notes
Normal serum	Serum is typically diluted to a final concentration of 5-10 % in PBS-T. In indirect detection, serum should be from the same species as the host of the secondary antibody or in the case of direct detection, goat serum can be used.
BSA	BSA is commonly dissolved to a final concentration of 1-5 % in PBS-T. It is important to use BSA, free of endogenous IgG molecules as they tend to cross-react with secondary antibodies and cause an increase in background signal.
Milk Powder	Non-fat milk powder is dissolved in PBS-T to a final concentration of 1 %. Blocking with milk is not recommended for detection of phosphorylated proteins due to the high content of phosphoproteins which can interfere with staining and contribute to high-background staining.