A retrograde double-labeling technique for light microscopy. A combination of axonal transport of cholera toxin B-subunit and a gold-lectin conjugate.

A light microscopical, non-fluorescent, retrograde double-labeling technique is described. Cholera toxin B-subunit (CTb) and a conjugate of wheatgerm agglutinin and bovine serum albumin coupled to 10 nm gold particles (gold-lectin) are both excellent retrograde tracers and, when visualized by means of immunohistochemistry and silver intensification, respectively, may be readily identified within the same cell. This light microscopical retrograde double-labeling technique is illustrated in rat with experiments designed to investigate the collateralisation (1) of vestibular neurons to the spinal cord and oculomotor complex, (2) of spinal neurons to the left and right lateral reticular nucleus, and (3) of inferior olivary neurons to the uvula of the cerebellum. Advantages over fluorescent double-labeling experiments are found in the fact that the diaminobenzidine reaction product as well as the silver/gold deposits do not fade and can be examined in counterstained sections. Moreover, the injection sites can be kept quite small and may be guided by electrophysiological recording through the injection pipette.