Sensitive double-labeling technique of retrograde biotinized tracer (biotin-WGA) and immunocytochemistry: light and electron microscopic analysis.

We developed a simple method to identify fiber projections that contain bioactive substances using biotinized wheat-germ agglutinin. Tracer that accumulated retrogradely in the soma was made visible under the light microscope by linking it with streptavidin-Texas red, which has a red fluorescence, while antigen was demonstrated in the same section by indirect immunofluorescence using fluorescein isothiocyanate with green fluorescence as a marker. Under the electron microscope, the tracer was identified using streptavidin-colloidal gold particles, while the antigen was demonstrated in the same ultrathin section using the peroxidase-antiperoxidase technique. This method makes it possible to demonstrate the locations of both the biotinized retrograde tracer and the antigen in the soma of the same neuron at both light and electron microscopic levels.