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, nan-fluorescent, retrograde double-labeling tec hnique is described. Cholera toxin B-subunit (CTb) and a conjugate of wheatgerm agglutinin and bovine serum albumin coupled to 10 nm gold pa rticles (gold-lectin) are both. excellent retrograde tracers and, when visualized by means of immunohistochemistry and silver intensificatio n, respectively, may be readily identified within the same cell. This light microscopical retrograde double-labeling technique is illustrate d in rat with experiments designed to investigate the collateralisatio n (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 t he fact that the diaminobenzidine reaction product as well as the silv er/gold deposits do not fade and can be examined in counterstained sec tions. Moreover, the injection sites can be kept quite small and may b e guided by electrophysiological recording through the injection pipet te.