Fig. 7.

Double immunolabeling of the innervation to the epidermis and upper dermis in 7-week-old mice (A–D) and single labeling in P2 mice (E–G). The broken line box in A is a PNC which is shown enlarged in Figure 9. Red symbols indicate Cy3 labeling only against the antigen noted on the images in the first column and green symbols indicate AlexaFluor 488 only against GFP or β-gal (second column of images). Yellow symbols indicate double labeling as confirmed in the merged images of the third column. Arrows are miscellaneous nerves and profiles in the upper dermis. Arrowheads are endings in the epidermis. A: GFP immunofluorescence is present in much (yellow symbols), but not all of the innervation (red symbols) that is labeled with anti-PGP. B: β-gal can be detected on some axons (yellow arrows) in the upper dermis, but most axons and all the epidermal endings lack β-gal (red arrows and arrowheads, respectively). C: CGRP and GFP immunolabeling is mostly separate (red and green arrows), although separately labeled processes can be closely intertwined. Only a few profiles are truly double labeled (yellow arrows). D: NF200 and GFP immunolabeling is highly coextensive. The thicker NF200/GFP-positive fibers (larger yellow arrows) are presumptive Aδ fibers that have previously been shown to lack substance P (SP) and CGRP. The thinner NF200/GFP-fibers (smaller yellow arrows) are presumptive Aδ fibers that have been previously shown to contain SP and CGRP. A few fibers only label for GFP (green arrows) and are presumably a type of C-fiber innervation. E–G: The arrows indicate innervation at comparable locations to the upper dermis and epidermis at P2. Of the total innervation revealed by anti-PGP (E), a large proportion expresses immunoreactivity for TrkA (F) and a smaller proportion for TrkC (G). Much of the innervation does not label for either receptor. Scale bars = 50 μm.