Is my antibody‐staining specific? How to deal with pitfalls of immunohistochemistry

Immunohistochemistry is a sensitive and versatile method widely used to investigate the cyto‐ and chemoarchitecture of the brain. It is based on the high affinity and selectivity of antibodies for a single epitope. However, it is now recognized that the specificity of antibodies needs to be tested in control experiments to avoid false‐positive results due to non‐specific binding to tissue components or recognition of epitopes shared by several molecules. This ‘Technical Spotlight’ discusses other pitfalls, which are often overlooked, although they can strongly influence the outcome of immunohistochemical experiments. It also recapitulates the minimal set of information that should be provided in scientific publications to allow proper evaluation and replication of immunohistochemical experiments. In particular, tissue fixation and processing can have a strong impact on antigenicity by producing conformational changes to the epitopes, limiting their accessibility (epitope masking) or generating high non‐specific background. These effects are illustrated for an immunoperoxidase staining experiment with three antibodies differing in susceptibility to fixation, using tissue from mice processed under identical conditions, except for slight variations in tissue fixation. In these examples, specific immunostaining can be abolished depending on fixation strength, or detected only after prolonged postfixation. As a consequence, antibody characterization in immunohistochemistry should include their susceptibility towards fixation and determination of the optimal conditions for their use.