Sc 101820

The eyeballs were fixed at 4 °C in PBS-buffered 4% paraformaldehyde for 24 h, and the anterior segment of the eyeball, including the cornea, iris and lens, was dissected under microscopy. The rest of the eyecup was dehydrated in 30% sucrose solution for 2 h and embedded in optimal cutting temperature compound for cryosectioning. Sections (10 μm) were cut on a Leica microtome (German) and mounted on adhesion microscope slides (Citoglas Company, Taizhou, China). Slides with sections were dried for 4 h at room temperature.
For immunostaining, the sections were incubated in PBS for 10 min; then, they were permeabilised in 0.25% Triton X-100 for 10 min. After washing thrice in PBS, the sections were blocked with 1% BSA for 30 min; then, they were incubated overnight at 4 °C with primary antibodies, i.e., HIF-1alpha (1:100, MAB5382, Chemicon, USA), Flk-1 (SC-101820, Santa Cruz Biotechnology), VEGF (1:100, SC-152, Santa Cruz Biotechnology), EPO (1:500, ab65394, Abcam), GFAP (1:500, ab7260, Abcam) and GS (1:500, ab64613, Abcam). The sections without primary antibody served as negative control. After being washed for 15 min in PBS, the sections were incubated with the appropriate secondary antibody (1:100, anti-mouse FITC or anti-mouse CY3) for 1 h at room temperature in the dark. Then, the sections were coverslipped for examination under a fluorescent microscope.