Immunocytochemical detection of 5'-bromodeoxyuridine in fluoro-gold-labeled neurons: a simple technique to combine retrograde axonal tracing and neurogenetic characterization of neurons.

To characterize the axonal projections of 5'-bromodeoxyuridine (BrdU)-labeled neurons, we have combined retrograde tracer injection of Fluoro-Gold with the immunocytochemical detection of BrdU. Pregnant mice were labeled with pulses of BrdU at embryonic days E12, E13, E14, or E16. Young adult offspring were perfused with 4% paraformaldehyde 2 days after receiving a Fluoro-Gold injection into the cerebral cortex, thalamus, or hippocampus. Brain sections were processed for immunocytochemical visualization of BrdU using the peroxidase-anti-peroxidase method and a diaminobenzidine-nickel ammonium sulfate (DAB-Ni) reaction, and finally observed on a microscope equipped with brightfield and fluorescence optics. Both BrdU-immunoreactive nuclei and retrogradely labeled Fluoro-Gold-positive cells were detected. Double-labeled neurons were recognized by the presence of fluorescent particles in the cytoplasm and a black immunoreactive nucleus. Since both labelings occurred in different cell compartments, Fluoro-Gold granules were not obscured by the DAB-Ni precipitate. The method shown here permits a correlation of the neurogenesis of subsets of neurons identified by their BrdU content with the specific target into which such cells project.