Simultaneous observation of fluorescent retrogradely labeled neurons and the immunofluorescently labeled fibers apposing them using Fluoro-Gold and antisera labeled with the blue fluorochrome 7-amino-4-methylcoumarin-3-acetic acid (AMCA)

In this paper we describe a method allowing the simultaneous observation of fluorescent retrogradely labeled neurons and the immunofluorescently labeled fibers which appose them. This technique employs the fluorescent dye Fluoro-Gold to label neurons retrogradely, and 7-amino-4-methylcoumarin-3-acetic-acid (AMCA) conjugated to a secondary antiserum as a label for fluorescence immunohistochemistry. Both Fluoro-Gold and AMCA are excited by near-ultraviolet (UV) light, but under UV excitation Fluoro-Gold appears yellow and AMCA appears blue. Thus the two fluorochromes are both visible under a single condition of illumination and can be readily distinguished by color. This allows quick and accurate determination of whether or not immunofluorescent fibers appose retrogradely labeled neurons.