Fig. 6.

Double-labeling immunofluorescence with antibodies against CB₂, β-endorphin, and ETRB in the epidermis of glabrous skin from the hindpaw of a rat. The images are from sections that alternate with those in Fig. 5. Images are from locations indicted by the same letters in the schematic in Fig. 5. The dotted lines indicate the location of the basement membrane at the epidermal-dermal border. SC, stratum corneum; SL, stratum lucidum; SG, stratum granulosum; SS, stratum spinosum; SB, stratum basalis. (Scale bar, 50 μm; scale bar for insets, 25 μm.) (A and B) Anti-CB₂ labeling revealed with anti-rabbit Fab conjugated with Cy3, followed by anti-β-endorphin labeling revealed with anti-rabbit Ig conjugated with Alexa Fluor 488. CB₂ immunolabeling is mostly limited to stratum granulosum (red arrows), whereas β-endorphin immunolabeling spreads through stratum granulosum and into stratum spinosum (green arrows). Although fairly ubiquitously distributed throughout all areas of the epidermis, both labels can in parallel be restricted relatively superficially in some locations (*). B Inset is a 2× enlargement of the site in the small rectangle demonstrating the overlap of CB₂ labeling (yellow arrows) in stratum granulosum with β-endorphin, which also extends more deeply into stratum spinosum (green arrow). (C and D) Anti-ETRB labeling revealed with anti-rabbit Fab conjugated with Cy3, followed by anti-CB₂ labeling revealed with anti-rabbit Ig conjugated with Alexa Fluor 488. CB₂ labeling (between green arrows) is fairly uniformly distributed in all areas of the epidermis and overlaps with the restricted sites of ETRB labeling in the volar pads and patchy areas of ETRB labeling in flat skin surfaces (red arrows and arrowheads). D Inset is a 2× enlargement of the site in the small rectangle demonstrating some superficial to deep segregation among the overlap of CB₂ and ETRB labeling. The most superficial keratinocytes express primarily CB₂ (green arrowhead) and the deeper ones primarily ETRB (red arrowheads). Keratinocytes in between express both (yellow arrowheads).