Bisbenzimide: a fluorescent counterstain for tissue autoradiography

Interpretation of the data from experiments using autoradiography (e.g. using in situ hybridization histochemistry, receptor binding, neuronal tract-tracing etc.) is aided when the autoradiographic grains can be seen in the context of cellular boundaries. Studies making use of autoradiography in the central nervous system have sometimes used tinctorial stains, such as cresyl violet, as counterstains to visualize the labeling. Tinctorial stains are excellent Nissl stains however, under bright-field illumination such dyes tend to obscure autoradiographic grains. In addition, dark-field illumination provides a common means of visualizing autoradiographic grains but tictorial stains are not optimally visible under these conditions. In an effort to find a counterstain that would be compatible with dark-field illumination, we have investigated the use of fluorescent dyes. Of the fluorescent dyes tested, bisbenzimide (Hoechst 33258) in pH 2.0 buffer was found to be optimal. Bisbenzimide counterstaining gave good resolution of cellular boundaries and appeared not to interfere with the ability to visualize autoradiographic grains. Furthermore, the illumination of bisbenzimide and of the autoradiographic grains could be controlled independently, making it easy to visualize or photograph the bisbenzimide Nissl staining and the autoradiographic grains simultaneously. Thus, bisbenzimide is well suited for use as a fluorescent counterstain in autoradiographic studies.