Simultaneous labeling of projecting neurons and apoptotic state

We describe a straightforward method that accomplishes both the labeling of projecting neurons and the identification of apoptosis in those neurons. A single dye, 4′,6-diamidino-2-phenylindole (DAPI), is both retrogradely transported and binds DNA. When delivered to the sites of neuronal projections, DAPI travels via retrograde transport from neuronal projections to the soma and stain nuclei with little or no cytoplasmic labeling. The staining of the nuclei allows for visualization of their morphological characteristics; DAPI-stained living cells appear markedly different from DAPI-stained apoptotic cells, due to the nuclear changes that apoptotic cells undergo. This technique has been successfully employed with retinal ganglion cells in the retinocollicular pathway. The use of a single dye not only eliminates the need for secondary staining for apoptosis, but also allows for the use of a wider variety of non-overlapping fluorescent dyes for other studies.