Embryonic Corneal Wound Healing Imaging

Embryos with wounded corneas were collected at 3, 5, 8, 9, 10 and 11 days post wounding (dpw). Following decapitation, eyes were collected in Ringer’s solution and fixed overnight in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), pH 7.2. Corneas were dissected from the surrounding scleral tissue, mounted on glass coverslips with 50% glycerol in PBS (v/v) and imaged en face for whole-mount imaging. For cross-section imaging, corneas were embedded in 10% low melting point agarose (NuSieve GTG; Lonza, Rockland, ME, USA) as previously described^{52 (link)}. A schematic representation of the wounded embryonic cornea, the tissue orientation, and imaging approaches is illustrated in Fig. 1. Tissue sections of approximately 300 µm thick were cut using a vibratome (Campden Instruments Ltd.) and imaged. The wounded corneas were grouped according to the different phases of the wound healing process; early (3–5 dpw), mid (8–9 dpw) and late (10–11 dpw) healing. At least three wounded and unwounded (control) corneas were analyzed in each group.