Antibody diluents are very important to the IHC process. The wrong mixture can prevent the optimal antibody-antigen connection. Diluents are made up of Tris HCl, detergents, stabilizers, serum, bovine serum albumen (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 itself to stabilize/preserve the antibody so additional sodium azide in the diluent is not necessary and may reduce antibody activity. The type of buffer used in the diluent should be used throughout the staining procedure for reagent consistency and phosphate buffered saline alone should not be used as a diluent.