Figure 7. Confocal double-label immunocytochemical localization of Mel1a and ZO-1 in Xenopus corneal whole mounts. A: Mel1a and ZO-1 immunolabeling is observed on the lateral plasma membrane, with some immunoreactivity also occurring in the cytoplasm. Mel1a (red) labeling is present predominantly as very broad bands on the lateral membranes, including the obliquely-oriented lateral membranes (arrows). The plasma membrane has an abundance of yellow labeling, indicative of a very close proximity of red Mel1a and green ZO-1. The ZO-1 labeling was not as broadly distributed on the lateral membrane. 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. Arrows are provided as reference points to indicate the same points on panel A. The rotated image shows that the green ZO-1 labeling is generally located apically to the red Mel1a labeling. The ZO-1 labeling appears as a relatively narrow continuous band of green labeling on the lateral plasma membrane, whereas a much broader band of red Mel1a labeling appears directly basal to the ZO-1 label. Significant yellow labeling is observed, indicating that the Mel1a receptor is in very close proximity to ZO-1. The confocal images in both panels are comprised of seven optical slices of 400 nm each in the z-series. The magnification bar (B) represents 20 µm.