To study the developing murine retinal vasculature and hyaloid system, neonatal C57-BL/6J mice were killed on postnatal days (P)1, P3, P5, P7, P9, and P11 after birth. Before death, some mice were heavily anesthetized (Hypnorm; Janssen-Cilag, High Wycombe, UK; 0.01 mL/g intraperitoneally; fentanyl, 0.315 mg/mL; fluanisone, 10 mg/mL; and midazolam, 5 mg/mL) and perfused with fluorescein isothiocyanate (FITC)-dextran (molecular weight 2 × 106; Sigma Chemical Co., Poole, UK), dissolved in phosphate-buffered saline (PBS), to assess the extent of retinal vascular development. After enucleation, the eyes were fixed in 4% paraformaldehyde (PFA) for 4 hours and then placed in PBS at 4°C. The anterior segment, lens, vitreous, and hyaloid were removed, and the posterior eye cup was subjected to four radial full-thickness cuts to facilitate flat mounting. FITC-dextran-perfused eyes and labeled eyes were viewed by confocal scanning laser microscopy (CSLM; MicroRadiance; Bio-Rad, Herts, UK) after they were flat mounted onto microscope slides. Eyes to be processed for immunohistochemistry and in situ hybridization were enucleated in mice under terminal anesthesia and fixed in 4% PFA for 2 hours. After fixation, the eyes were washed in PBS before dehydration and embedding in wax for standard histologic sectioning.
Treatment of the mice throughout this study was in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and according to British governmental guidelines.