Differential labeling of converging afferent pathways using biotinylated dextran amine and cholera toxin subunit B

We report a new technique for 2-tracer anterograde labeling that permits unequivocal identification of the differentially labeled projections in the same section. One pathway is labeled with biotinylated dextran amine and is visualized as a black to dark gray diaminobenzidine (DAB)-cobalt precipitate by an avidin-biotinylated peroxidase reaction. The other pathway is labeled with cholera toxin subunit B and is visualized as a reddish-brown reaction product using DAB without cobalt as the substrate for peroxidase immunohistochemistry. To maintain serial order, sections can be processed mounted on slides without any loss of sensitivity for either tracer.