Figure 6.

Three-color imaging with NV Jade, Red, and Maroon allows excellent resolution of thin fibers and discrete nerve tracts in near-adult murine brain. Nerve tracts projecting from one region of mature brain to another were labeled with NV Jade (green pseudocolor), NV Red (red pseudocolor) or NV Maroon (blue pseudocolor) and imaged by confocal microscopy after 5 days of diffusion at 37 C. Bar indicates 100 μm in all images. Panel A: Despite increased green autofluorescence due to myelinization of near-adult brain, NV Jade can be used in 18-day-old cerebella to label not only parallel fibers (green lines crossing from top left to bottom right) but also individual cells (stellar neuron in the center of A) at distances of up to 5 mm away from the injection site. (Representative image selected from 12 replicate specimens.) Panels B and C: After labeling in the pontine nuclei, cerebellar cortex and restiform body of the cerebellum with NV Jade, NV Red or NV Maroon, respectively, discrete fibers are consistently labeled and readily visualized near the deep cerebellar nuclei of P18 animals without bleed through and at distances of up to 1 cm away from the injection site over 50 animals. The same type of labeling was used on two replicate specimens, which were cut slightly differently to show fiber bundles running in parallel (B) or crossing one another (C).