Quantitative immunohistochemistry in the rat facial nucleus with [125I]-iodinated secondary antibodies and in situ autoradiography: non-linear binding characteristics of primary monoclonal and polyclonal antibodies.

Abstract

Indirect immunohistochemistry is an important routine method in histology and histopathology. Here we have investigated the quantitative aspects of antibody binding to tissue sections, using a range of monoclonal and polyclonal antibodies and the regenerating rat facial nucleus as the experimental model. The in situ binding of primary antibodies was quantified using appropriate [125I]-iodinated secondary antibodies and quantitative autoradiography. The majority of primary antibodies revealed an apparently bell-shaped curve of in situ antibody binding, with the binding increasing up to a specific antibody concentration and then decreasing; similar data were also obtained with enzymatic immunohistochemistry. There was also a close correlation between the quantitative changes in antibody binding during the time course of facial nerve regeneration and those observed with enzyme histochemistry.