Fluorescent double-labeling with carbocyanine neuronal tracing and immunohistochemistry using a cholesterol-specific...

The fluorescent carbocyanine dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) has been widely used for tracing of neuronal pathways. To examine identities of the DiI-labeled neuronal pathways, it is desirable to combine DiI labeling with immunofluorescent staining. However, DiI labeling and immunofluorescent staining are not well compatible, mainly because treatment of DiI-labeled neurons with detergents, which are commonly used for immunohistochemistry, results in high levels of diffusion of the DiI label. In this study, we searched for detergents that are compatible with DiI labeling, and found that a cholesterol-specific detergent digitonin is useful for fluorescent double-labeling with DiI tracing and immunohistochemistry. We show that digitonin treatment, in contrast to Triton X-100, methanol and Nonidet P-40 treatment, preserves DiI labeling, even at higher concentrations. We also show that digitonin also preserves the signal of a DiI derivative CM-DiI. Moreover, we demonstrate that digitonin efficiently increases antibody penetration into brain sections. As a result, immunohistochemical images obtained with digitonin treatment are as good as those obtained with Triton X-100 treatment. In addition, we also try another cholesterol-specific detergent quillaja saponin, but find that it degrades the DiI label. Our simple double-labeling protocol using digitonin should prove useful in enabling detailed examination of the neuronal circuitry of the nervous system.