Figure 3. Confocal double-label immunocytochemical localization of Mel1a and Mel1c in Xenopus corneal whole mounts. A: The specimen shown was obtained in the mid-light period (12N). Both Mel1a and Mel1c immunolabeling is observed on the lateral plasma membrane, with some immunoreactivity also occurring in the cytoplasm. The labeling of the plasma membrane displays distinct areas of Mel1a (red), Mel1c (green), and both receptors (yellow). Arrows are provided as reference points to indicate the same points on 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 red Mel1a labeling is generally located apically to the green Mel1c labeling. The Mel1a labeling is seen as a relatively broad continuous band of red label on the lateral plasma membrane of the majority of surface CE cells. A somewhat broader band of green Mel1c labeling appears directly basal to the Mel1a label. Some yellow labeling is occasionally observed, indicating some co-localization of Mel1a and Mel1c. There are many areas in the red Mel1a band in which yellow labeling is interspersed between areas of red Mel1a labeling, suggesting that some green Mel1c-labeled receptor is interdigitated among the Mel1a-labeled receptor. The confocal images in both panels are comprised of 13 optical slices of 400 nm each in the z-series. The magnification bar (B) represents 20 µm.