Using the primary antibody at the correct dilution is essential for optimal staining results. Antibody diluents are protein-based, buffered solutions used to dilute concentrated antibodies. They can comprise Tris HCl, detergents, stabilizers, serum, bovine serum albumin (BSA), or other proteins. Diluents should be buffered to physiological pH (pH = 7.0–7.2 preferably, but can be up to pH 7.4) to control ionic interactions between antigens and antibodies.
Considerations for the ideal antibody diluents include:
- Does it contain normal serum?
- Does it contain hazardous substances such as sodium azide?
- What is the buffer base?
Normal serum can bind with secondary antibodies and/or reduce sensitivity. Sodium azide is usually found in the antibody to stabilize/preserve the antibody, so additional sodium azide in the diluent is unnecessary and may reduce antibody activity. The type of buffer used in the diluent should be used throughout the staining procedure for reagent consistency. Phosphate-buffered saline (PBS) alone should not be used as a diluent.
