A retrograde double label tracing technique using horseradish peroxidase and the fluorescent dye 4'-,6-diamidino-2-phenylindole 2HC1 (DAPI).

Retrograde transport of HRP and the fluorescent dye DAPI were used to double label spinothalamic tract cells with dual projections to thalamus and cerebellum. Cells containing both retrograde tracers were found in the intermediate gray zone and in the lateral spinal nucleus of the rat lumbosacral spinal cord. Double labeled cells were easily identified by the presence of granular HRP reaction, product and the blue fluorescence of DAPI. The use of HRP and DAPI provides an effective double labeling technique for the localization of neurons with nonoverlapping projections in the central and/or peripheral nervous system.