To label cells that are in the S-phase of the cell cycle, mice were injected once intraperitoneally with BrdU (150 mg/kg) and were killed after periods ranging from 2 hours to 21 days. Corneas were removed from the enucleated eyes, and the eyecups were fixed in 4% paraformaldehyde (PFA) for 2 hours at room temperature followed by cryoprotection in 30% sucrose overnight at 4°C. We embedded the eyecups in OCT compound (Tissue-Tek; Sakura Finetek, Tokyo, Japan) and cut 12-μm frozen sections through the dorsal to ventral meridian.
Sections were stained with primary antibodies for rhodopsin (1:1500; Chemicon, Temecula, CA), VEGFR1/Flt1 (1:30; R&D Systems, Minneapolis, MN), VEGFR2/Flk1 (1:30; R&D Systems), Pax 6 (1:1000; Developmental Studies Hybridoma Bank, Iowa City, IA), or BrdU (1:1000 [Developmental Studies Hybridoma Bank] and 1:1000 [Oxford Biotechnology, Raleigh, NC]), followed by Alexa 488-, 568-, or 647-conjugated secondary antibodies (all at 1:1500; Molecular Probes, Eugene, OR). A TUNEL assay kit (Roche, Basel, Switzerland) was used, in accordance with to the manufacturer's instructions, to detect apoptotic cells.
Next the flat mount specimens were subjected to BrdU-staining, as follows: after the PFA-fixed eyecups were flattened with radial incision, they were permeabilized in 0.5% phosphate-buffered saline Triton X-100 (PBST) for 2 hours, incubated in 2 N HCl for 45 minutes, and neutralized with 0.1 M Na2B4O7 for 15 minutes. After blocking with 5% goat serum in PBS for 1 hour, the eyecups were incubated with anti–BrdU antibodies for 12 hours and with secondary antibody for 9 hours.