Fig. 2. Confocal micrographs of BrdU and TOTO-1 labeling in lens epithelial wholemounts 3 hours after injection of BrdU. Seven-day-old wild-type or A^–/– mice were injected with BrdU and wholemounts of lens epithelium were fixed 3 hours after the BrdU injection. The chromosomes were stained with BrdU. (A,D) S-phase cells labeled with BrdU (red) in wild-type (A) or A-knockout (D) lenses. (B,E) Cells labeled with the DNA stain TOTO-1 in the same wholemounts (green): (B) wild-type; (E), A^–/–. (C,F) Merged confocal images of BrdU and TOTO-1: (C) wild-type; (F) A^–/–. Note that the majority of the BrdU-labeled cells are single cells. A pair of mitotic cells can be seen in TOTO-1 staining of the wild-type epithelial wholemount (arrowheads). The nuclei of the lens epithelium demonstrate several staining patterns, including uniform light labeling, punctate labeling and uniform intense labeling. Note also that the first labeled mitoses were observed 3 hours after BrdU injection (A-C). (G) Quantitative analysis of the labeling index in the wild-type and A^–/– wholemounts 3 hours after the BrdU pulse. Note that 3 hours after the BrdU injection, the labeling index was the same in wild-type and A^–/– epithelial cells. Also note that the minor differences seen in the nuclear size of the wild-type and A^–/– wholemounts were not consistently observed, and show variation in the spreading out and fixation during preparation of the wholemounts. Bars, 25 µM (A-C); 25 µM (D-F).