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The page below is a sample from the LabCE course Immunohistochemistry (IHC) Basics in Histology. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Antibody Validations

Any antibody brought into the laboratory must be validated to ensure reproducibility and consistency in results. Each antibody may have its own unique protocol, but it is important to test it under the recommended conditions in order to provide the best results. The antibody specification sheet that accompanies all commercially prepared antibodies will provide a recommended protocol. After a control is selected, the tissue must be tested on the recommended protocol for at least a place to begin validation. The antibody manufacturer recommended protocol may or may not produce the best result in your laboratory. It is wise to test a few protocols to see which protocol actually provides the best staining.
The important factors to consider in validating an antibody protocol are:
  • Antigen retrieval (pretreatment solution and time)
  • Enzyme pretreatment
  • Antibody incubation time
Pre-diluted antibodies are easy to use and fairly easy to 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 dilutions and validate over a series of dilutions.
If attempted validation 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 put in practice and tested on any patient tissue. Appropriate documentation of this process should be part of the QC in your laboratory.