Anterograde axonal tracing with the subunit B of cholera toxin: A highly sensitive immunohistochemical protocol for revealing fine axonal morphology in adult and neonatal brains

We report an improved immunohistochemical protocol for revealing anterograde axonal transport of the subunit B of cholera toxin (CTB) which stains axons and terminals in great detail, so that single axons can be followed over long distances and their arbors reconstructed in their entirety. Our modifications enhance the quality of staining mainly by increasing the penetration of the primary antibody in the tissue. The protocol can be modified to allow combination in alternate sections with tetramethylbenzidine (TMB) histochemical staining of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Using this protocol, we tested the performance of CTB as an anterograde tracer under two experimental paradigms which render other anterograde tracers less sensitive or unreliable: (1) labeling the entire retinofugal projection to the brain after injections into the vitreal chamber of the eye, and (2) labeling developing projections in the cortex and thalamus of early postnatal mammals. Qualitative comparisons were made with other tracers (Phaseolus vulgaris leucoagglutinin, dextran rhodamine, biotinylated dextran, free WGA, or WGA-HRP) that were used to label these same projections. From these observations it is clear that CTB, visualized with our protocol, provides more sensitive anterograde labeling of retinofugal projections as well as of axonal connections in the neonatal forebrain.