Any antibody brought into the laboratory must be optimized and validated to ensure reproducibility and consistency in results. Primary antibodies should be optimized before validation. Optimization includes finding the appropriate dilution (for concentrated antibodies), optimal antigen retrieval method, and antibody incubation time. The antibody product insert sheet contains recommendations for the starting point of optimization. Optimization control tissues 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 relatively easy to optimize and validate, but they do not offer the most extended shelf life or lowest cost. Purchasing concentrated antibodies is more cost-effective, but it takes more time to perform antibody titrations and optimize over a series of dilutions.
If an antibody optimization attempt fails, change only one variable at a time, such as retrieval or antibody incubation, to proceed efficiently and find the best method for optimal staining. Also, when a new lot of a validated antibody is received, it must be revalidated with the latest lot, and the results must be compared to the previous lot to ensure appropriate staining before being put into practice and tested on any patient tissue. Proper documentation of this process should be part of the QC in your laboratory.
