Any antibody brought into the laboratory must be optimized and then validated to ensure reproducibility and consistency in results. Primary antibodies should be optimized prior to validation. Optimization includes finding the appropriate dilution (for concentrated antibodies), optimal antigen retrieval method, and antibody incubation time. The antibody product insert sheet includes recommendations for the starting point of optimization. Control tissues used in optimization should be fixed and processed identically to patient tissue.
After antibody optimization is complete, validation of selected staining parameters is achieved by performing IHC staining with the new antibody on a set number of slides that include tissue sections with a wide range of reactivity ("strongly positive", "moderate to weak", etc.).
Pre-diluted antibodies are easy to use and fairly easy to optimize and validate but do not offer the longest shelf life or lowest cost. Purchase of concentrated antibodies are more cost effective, but it takes more time to perform antibody titrations and optimize over a series of dilutions.
If attempted optimization of an antibody fails, change only one variable at a time, such as retrieval or antibody incubation, to proceed in order to efficiently find the best method for optimal staining. Also, when a new lot of a validated antibody is received, it must be revalidated with the new lot and the results compared to the previous lot to ensure appropriate staining before being put in practice and tested on any patient tissue. Appropriate documentation of this process should be part of the QC in your laboratory.
