The present report deals with a multiple tract-tracing procedure in non-human primates enabling the simultaneous visualization of retrogradely transported Fluoro-Gold (FG) and cholera toxin B subunit (CTB) in combination with anterogradely transported biotinylated dextran amine (BDA). Two issues have played key roles on the achievement of this reliable procedure: first, the recent development of a commercial antiserum against FG that allows us to convert the original fluorescent signal of this dye in a permanent precipitate via standard peroxidase-anti-peroxidase methods; second, the introduction of the novel peroxidase substrate Vector(R) VIP (V-VIP), resulting in a purple precipitate. The combination of these neuroanatomical tracers in one and the same histological section opens a possibility for the permanent visualization of the convergence of inputs from a particular brain area onto identified, two different subsets of projection cells of another area. Furthermore, this combination of three tracers emerges as a powerful technical tool for obtaining broad amounts of complementary data regarding the monkey brain connectivity, thus significantly reducing the number of animals needed to complete a particular study.
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