Double-staining of CGRP and synaptophysin or IB4. (A) Double-staining of CGRP (red) and synaptophysin (green) in rat C1. (B) Synaptophysin immunoreactivity is found all laminae, with fibers extending into to the white matter. Circular immunoreactive fiber formations are seen around neurons (arrows). (C) Double-staining of CGRP (green, thin arrows) and IB4 (red, thick arrow). IB4 is mostly expressed in laminae II/III. IB4 is also expressed in laminae I/II which could be seen as co-localization with CGRP. However, confocal microscopy (D) revealed that CGRP (green, thin arrow) and IB4 (red, thick arrow) are most often not expressed in the same fibers. Occasionally the two markers co-localize in fibers (arrow head).