Specificity Controls in Immunohistochemistry

Immunohistochemistry is a powerful method for identification of proteins in cells and tissues, but control procedures are necessary. The specificity of the results depends on two independent criteria the specificity of the antibody and of the method used (Burry 2000). The antibody specificity is best determined by immu-noblot and/or immunoprecipitation. The specificity of the method is best determined by both a negative control, replacing the primary antibody with the corresponding IgG (must be the same species as primary antibody at the same concentration), and a positive control, testing the primary antibody with tissues known to contain the antigen. 

An additional control for the antibody specificity is the so-called absorption or preabsorption control, in which the primary antibody (prior to its use) is incubated for 1 h with a tenfold molar excess of the purified antigen. Absent or greatly diminished immunostaining should be obtained after application of this preabsorbed antibody. However, it is sometimes difficult to obtain the purified antigen therefore, it is rarely used routinely in immunohistochemical staining. Moreover, absorption of the antibody with the purified antigen does not always indicate that the antibody has bound to the same protein in the tissue (Burry 2000). 

When fluorescent dyes are used in the experiments, autofluorescence (or natural fluorescence) of some tissue components can cause background problems and complicate the use of fluorescence microscopy. The simplest test is to mount 

Burry RW (2000) Specificity controls for immunocytochemical methods. J Histochem Cytochem 48 163 166 

Lipman NS, Jackson LR, Trudel LJ, Weis Garcia F (2005) Monoclonal versus polyclonal antibodies distinguishing characteristics, applications, and information resources. ILAR J 46 258 268 

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