Three-color immunofluorescence histochemistry allowing triple labeling within a single section.

We describe a procedure for simultaneous immunohistochemical localization of three different neuropeptides, neurotransmitters, or neurotransmitter enzymes within one and the same tissue section and present a number of examples of its application within the brain and periphery. Primary antibodies from three different species were bound to three different neurochemical substances within the same section and were then reacted with three appropriate species-specific antisera conjugated with fluorescein, rhodamine/Texas red, or biotin. The biotinylated secondary antiserum was subsequently reacted with diethylaminocoumarin (DAMC) conjugated to avidin. This combination resulted in green, red, and blue fluorescent labeling of each antigen, respectively. Each fluorescent marker was viewed and photographed discretely using appropriate excitation and suppression filter combinations. The method is well suited for analyzing instances of multiple coexistence at both the level of the cell soma and within terminal regions. More broadly, the feasibility of three-color immunofluorescence histochemistry extends the range with which antigen localization can be used to investigate the morphological bases of relationships and interactions between immunohistochemically characterized neuronal elements.