The long-wavelength tracer nuclear yellow is often combined with the popular retrograde tracer true blue for two-color neuronal mapping. In neuronal cells, nuclear yellow primarily stains the nucleus with yellow fluorescence, whereas true blue is a UV light–excitable, divalent cationic dye that stains the cytoplasm with blue fluorescence. Both nuclear yellow and true blue are stable when subjected to immunohistochemical processing and can be used to photoconvert DAB into an insoluble, electron-dense reaction product. Bisbenzimide and Nuclear Yellow, when transported retrogradely through axons to their parent cell bodies, may migrate out of the axons and the cell bodies, as indicated by fluorescence of adjoining glial nuclei. This migration was found to take place both in vivo and in vitro during storage of the sections in water. When using the tracers in 1% concentrations the in vivo migration may be controlled by restricting the survival time. The in vitro migration may be prevented by rapid histological processing of the material.
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