Simultaneous immunocytochemical visualization of bromodeoxyuridine and neural tissue antigens

5'-Bromodeoxyuridine (BrdU) is a thymidine analogue which can be detected by monoclonal antibodies (MAb). We have developed a method for the simultaneous visualization of BrdU and a wide range of neural antigens in paraformaldehyde-fixed brain sections. Pregnant mice were injected intraperitoneally with a single pulse of BrdU. Young adult offspring were processed for immunocytochemistry following a double immunoperoxidase sequence. BrdU was detected using diaminobenzidine (DAB) intensified with nickel ammonium sulfate and neural antigen-containing elements were visualized with DAB alone. BrdU-positive nuclei and tissue antigen-immunoreactive cells were easily differentiated. Furthermore, double-labeled cells characterized by the presence of a black immunoreactive nucleus surrounded by a brown immunopositive cytoplasm were unambiguously recognized. Satisfactory results were obtained using either MAb or polyclonal antibodies against a variety of cell antigens, including neuropeptides, CA++ binding proteins, and cytoskeletal components of the glial cells. The method reported here permits analysis of the neurogenesis and proliferation of subsets of neurons and glial cells, identified by immunocytochemical markers.