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Fig. 2. Proliferation and differentiation of retinal cells perturbed by the ß-catenin signaling pathway. (A,B) BrdU incorporation into retinal cells expressing EGFP, caß-catenin-EGFP, caLEF-EGFP or dnLEF-EGFP was examined. Retinal explants were infected with retrovirus and incubated with 5 µM BrdU at 2, 6 or 13 days after retrovirus infection and harvested 24 hours later. (A) Patterns of frozen sections immunostained with anti-BrdU and anti-GFP antibodies were visualized with secondary antibodies conjugated to Alexa 546 and Alexa 488 (Molecular Probes), respectively. (B) Percentage of BrdU-incorporated retinal cells in virus-infected cells. (C,D) Expression of rod photoreceptor and Muller glia markers was examined by immunohistochemistry. Retinal explants were infected with retrovirus and cultured for 2 weeks. Frozen sections were then made (C), or the explants were disaggregated with trypsin and replated into chamber slides (D). In D, the number of Rho4D2-positive cells were counted. More than 100 cells were measured for each sample and s.d. was calculated from three independent experiments. Both samples were subjected to immunohistochemistry for rod photoreceptor cells by using anti-Rho4D2 antibody and for Muller glia cells by using anti-GS antibody. These first antibodies were visualized by using secondary antibody conjugated to Alexa 546. Nuclei were stained with DAPI and infected cells were detected by anti-GFP antibody. Bars, 50 µm (A,C).
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