Fig. 4. Merged confocal micrographs of BrdU- and TOTO-1-stained nuclei in the lens epithelial wholemounts. (A) In this micrograph, several BrdU-labeled `tetrads' can be seen (arrows) 24 hours after BrdU injection. Pairs of BrdU-labeled nuclei (red) were identified by their identical staining pattern. The wholemount was stained with TOTO-1 (green) to detect all the nuclei. Note that a majority of the nuclei were not labeled with BrdU. Bar, 25 µM. (B) BrdU and TOTO-1 staining in lens epithelial wholemounts 5 days after injection. Wild-type mice were injected with BrdU and wholemounts were fixed 5 days later. BrdU immunofluorescence (red) in the central region of the wholemount is shown. TOTO-1 (green) was used to label the nuclei of all the cells. Note that the BrdU-labeled cells were all members of cell pairs (arrows). Note also that, in some cases, members of a pair of BrdU-labeled nuclei were separated by two or more nuclei. Bar, 10 µM. (C) BrdU and TOTO-1 labeling in the periphery of a 7-day-old wild-type lens epithelial wholemount 24 hours after BrdU injection. Near the periphery (germinative region) of the lens epithelium, the labeling index was 2-3-fold higher as compared with the central region (Fig. 4A,B). In many cases, pairs of BrdU-labeled nuclei (red) that were very close to each other could be identified. In other cases, it was difficult to ascertain if two adjacent BrdU-labeled nuclei were members of a pair or not. TOTO-1 staining (green) was used to visualize all the nuclei. Bar, 25 µM.

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