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Fig.8. Xenopus rod aqueous spaces revealed by EGFP and confirmed by immunohistochemical labeling of cytochrome c and α-tubulin, and by EM. (A) Confocal image of transverse frozen section of retina with only the EGFP channel activated. Arrows point to bright dots that localize just below the OS, which align with a green line that projects up the OS. (B) Same section as in A, showing immunostaining of cytochrome c (red) and the nucleus with DAPI (blue). The green fluorescence in the same location as the DAPI labeling shows EGFP to be present in the nucleus. Note the presence of three cone nuclei (deep blue) which show no green labeling; these cones give rise to dark regions in the images of A and B. (C) Confocal image of transverse frozen section labeled with antibody against acetylated α-tubulin (red), which identifies the cilium and axonemes of two rods (arrows) and two cones (no arrows). (D) Same image as in C, but with the EGFP channel activated: the bright green dots are seen to localize at the projection of the axoneme to the top of the ellipsoid region. The EGFP channel is relatively less intense than in A and B because some of the protein is lost owing to the use of Triton to enable the antibody access to α-tubulin (Kaplan et al., 1987). When z-stacks of such confocal images are scanned, every single rod shows the features illustrated in these panels. (E-H) Montage of EM images of the ellipsoid and basal disc region of Xenopus rods: in these panels one can see a region devoid of mitochondria immediately surrounding the basal body of the cilium (arrows). Scale bars, 1 μm. |
