Diamidino yellow dihydrochloride (DY . 2HCl); a new fluorescent retrograde neuronal tracer, which migrates only very slowly out of the cell.

Earlier studies showed that Nuclear Yellow (NY), True Blue (TB) and Fast Blue (FB) are transported retrogradely through axons to their parent cell bodies. NY produces a yellow fluorescent labeling of the neuronal nucleus at 360 nm excitation wavelength, while TB and FB produce a blue fluorescence of the cytoplasm at this same wavelength. Therefore, NY may be combined with TB or FB in double-labeling experiments demonstrating the existence of axon collaterals. However, retrograde neuronal labeling with TB or FB requires a relatively long survival time, while NY requires a short survival time since NY migrates rapidly out of the retrogradely labeled neurons. This complicates double-labeling experiments since TB and FB must be injected first and NY later, a short time before the animal is sacrificed. We report a new yellow fluorescent tracer which labels mainly the nucleus and migrates much more slowly out of the retrogradely labeled neurons than NY. This new tracer can be used instead of NY in combination with TB or FB in double-labeling experiments and unlike NY can be injected at the same time as TB or FB. The new tracer is a diamidino compound (no. 28826) which is commercially available. It will be referred to as Diamidino Yellow Dihydrochloride (DY . 2HCl). According to the present study DY . 2HCl is transported over long distances in rat and cat, and produces a yellow fluorescence of the neuronal nucleus at 360 nm excitation wavelength, resembling that obtained with NY. When combined with TB or FB, DY . 2HCl is as effective as NY in double labeling of neurons by way of divergent axon collaterals.