Figure 5. Confocal double-label immunocytochemical localization of Mel1a and Mel1b in Xenopus corneal whole mounts. A: The specimen shown was obtained in the mid-light period (12N). Both Mel1a (red) and Mel1b (green) immunolabeling is present on the lateral plasma membrane appearing mostly as the merged yellow fluorescence indicative of co-localization. A significant amount of green Mel1b immunoreactivity is also present in the cytoplasm. Arrows are provided as reference points to indicate the same points on panel B. The inset illustrates the 72° rotation on the x-axis of the image in A, indicating the orientation relative to the viewer’s eye in B. B: Three-dimensional reconstructions of confocal z-stacks of optical slices were rotated at 72° degrees on the x-axis to enable optimal viewing of the pattern of immunolabeling. The rotated image shows that the Mel1a-Mel1b-labeled cells are characterized by a broad band of merged yellow labeling, interdigitating with a lesser amount of red Mel1a labeling. This pattern of labeling suggests that a majority of Mel1a and Mel1b receptors are located in very close proximity to each other on the lateral membrane. The confocal images in both panels are comprised of 19 optical slices of 400 nm each in the z-series. The magnification bar (B) represents 20 µm.