Anti islet 1 antibody

Retinal sections were obtained from mouse eyes fixed in 4% paraformaldehyde in PBS for 1 h on ice. The eyes were embedded in optimal cutting temperature (OCT) medium (Sakura Finetechnical, Torrance, CA) and snap frozen in liquid nitrogen. Frozen retinal sections were cut in a cryostat at 10 μm thickness along the vertical median through the optic nerve. Sections were rehydrated in PBS and blocked with blocking buffer (10% ChemiBlocker; Millipore, Billerica, MA) in PBS for 30 min at room temperature. Primary antibody rabbit polyclonal anti-Islet-1 antibody (Abcam, 1:250) was incubated in PBS containing 0.05% Tween 20 (PBTS) buffer overnight at 4 °C. The sections were washed with PBTS buffer five times (5 min each at room temperature). The slides were incubated with Alexa Fluor 488 anti-rabbit (Jackson ImmunoResearch, West Grove, PA; 1:200) 1 h at room temperature. The sections were washed with PBTS buffer five times (5 min each at room temperature) and rinsed with PBS. The chromosomal DNA was stained with 4’, 6-diamidino-2-phenylindole (DAPI; Roche diagnostics) and covered with a glass coverslip. Confocal fluorescence microscopy was performed using a Leica SP2. Images were subsequently processed using IMARIS (Bitplane Inc., Zurich, Switzerland) and Adobe Photoshop CS4 (Adobe Systems Inc).

The study protocol conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by the Ethics Review Committees for Animal Experimentation of Central South University. Small interference RNA (GIMA siRNA company, Shanghai, China). anti-Islet-1 antibody (Abcam , Cambridge, England),rabbit polyclonal anti-VEGF antibody (Abcam), Transwell (Corning Costar, Corning, NY), Matrigel (BD Biosciences, San Jose, CA), C57BL/6J mice (Shanghai Laboratory Animal Center, Shanghai,China).