Immunocytochemical controls

Riera J, Simpson JF, Tamayo R, et al. Use of cultured cells as a control for quantitative immunocytochemical analysis of estrogen receptor in breast cancer. The Quicgel method. Am. J. Clin. Pathol. 1999 111 329-335. 

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

As noted above, the specificity of the antibody-antigen reaction is critical for obtaining reliable, interpretable results. For this reason, the antibody has to be tested rigorously, and essential controls for antibody specificity should be included in any experimental design. A comprehensive discussion on antibody generation, specificity, and testing for immunocytochemical applications can be found in references (27-29) and, for specific applications, see Chapters 17, 50, and 51. 

Appropriate controls should always be run with any immunocytochemical procedure. Controls may include omitting the primary antibody, substituting pre-immune serum, normal serum, or normal IgG for the primary antibody, adsorbing the primary antibody against the antigen, or immunostaining with an unrelated antibody. 

The final application of the antibody must be borne in mind when deciding the extent of characterization. Initially, the antibody must be tested to establish whether binding occurs with the immunogen, with and without any carrier molecules used in the immunization. This test should be carried out with reference to the intended application to control for bridge binding. For instance, a reagent intended for use in a capture ELISA should be tested when coated onto the assay solid phase. If the antibody is intended for in vivo immunoneutralization, it should be tested initially in a liquid phase assay. If the final use is to be immunocytochemical, then the testing should be conducted on tissue sections. 

Butty RW. Specificity controls fot immunocytochemical methods. / Histochem Cytochem. 2000 48 163. 

Fig. 2. Effect of MDF on TH expression in cultures of E13 cerebral cortical cells. Photomicrographs are shown of cultures grow either on control media (a) or in the presence of MDF (b) overnight before being processed for the immunocytochemical localization of TH.

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