Corneal angiogenesis assay

The corneal micropocket assay was performed as described^37,38. Seven-week-old Akt1^WT and Akt1^∆SMC mice were anesthetized with chloral hydrate (450 mg/kg, i.p.). After 10 min, alcaine was dropped into the eye. A corneal micropocket was created with a modified von Graef knife and MVR knife in both eyes. A micropellet of sucralfate (Sigma-Aldrich) coated with hydron polymer (Sigma–Aldrich) containing 200 ng of VEGF was implanted into each corneal pocket. The pellet was positioned approximately 1 mm from the corneal lymbus. Seven days later, the mice were anesthetized with 1–2% inhaled isoflurane, and the eyes were captured using a digital camera. For staining, eyes were fixed with 4% paraformaldehyde for 12 h at 4 °C. The primary antibody was incubated in blocking buffer (3% BSA in PBS-Tween-20) overnight at 4 °C. The secondary antibody was diluted in blocking buffer and incubated for 2 h at room temperature. Corneas were flat-mounted using an anti-fading reagent, and images were obtained using a confocal microscope (K1-Fluo, Nanoscope Systems, Daejeon, Korea). Sprouting was quantified by measuring the VEGF-induced vessel sprouting length using ImageJ (National Institutes of Health).