Specificity Controls for Immunocytochemical Methods

TLDR

Immunocytochemistry localizes proteins in cells and tissues, but its specificity depends on both antibody quality and methodological controls, with antibody specificity best assessed by immunoblot or immunoprecipitation rather than absorption. The study emphasizes the need for appropriate controls to demonstrate labeling specificity as immunocytochemistry becomes more widely used. Method specificity is verified by a negative control (serum replacing the primary antibody) and a positive control using the antibody on cells known to express the target protein. J Histochem Cytochem 48:163-165, 2000.

Abstract

Immunocytochemistry is used for antibody localization of proteins in cells and tissues. The specificity of the results depends on two independent criteria: the specificity of the antibody and of the method used. The antibody specificity is best determined by immunoblot and or immunoprecipitation. Absorption of the antibody with a protein does not determine that the antibody would have bound to the same protein in the tissue, and therefore is not a good control for antibody specificity. The specificity of the method is best determined by both a negative control, replacing the primary antibody with serum, and a positive control, using the antibody with cells known to contain the protein. With the increasing use of immunocytochemistry, it is important to be aware of the appropriate controls needed to show specificity of the labeling. (J Histochem Cytochem 48:163-165, 2000)