Blocking buffers used in Immunohistochemistry (IHC-P) are substances used to reduce background staining and unspecific binding of the primary antibody to the tissue sections. Some commonly used blocking agents include normal serum (e.g. goat, sheep, or rabbit serum), BSA (bovine serum albumin), and non-fat dry milk. The choice of blocking buffer depends on the tissue type, the primary antibody used, and the desired staining outcome.
Bovine Serum Albumin (BSA), normal serum, and non-fat dry milk are all commonly used blocking agents in Immunohistochemistry (IHC), and each has its own advantages and disadvantages.
Benefits of BSA:
- High purity: BSA is a highly pure protein and does not contain other components that can interfere with the antibody-antigen reaction, making it useful for enhancing specificity.
- Consistent results: BSA is a highly uniform protein that provides consistent results between different batches and experiments, making it a reliable blocking agent.
Benefits of normal serum:
- Species specificity: Normal serum from the same species as the secondary antibody can enhance the specificity of the antibody-antigen reaction by reducing cross-reactivity with other proteins in the tissue.
- Cost-effectiveness: Normal serum is often a cost-effective alternative to BSA or non-fat dry milk.
Benefits of non-fat dry milk:
- Effective blocking: Non-fat dry milk contains proteins that can effectively block non-specific binding sites on the tissue sections, reducing background staining.
- Convenient: Non-fat dry milk is a ready-to-use blocking agent that does not require any additional preparation, making it convenient for some users.
Ultimately, the choice between these blocking agents depends on the tissue type, the primary antibody used, and the desired staining outcome. It's important to optimize the blocking conditions for each specific experiment.