Retinal Tissue Analysis in RCS Rats

After being anesthetized by 1% pentobarbital (150 mL/kg), RCS rats were perfused with normal saline and 4% paraformaldehyde via the circulation system on 3, 6, 9 and 12 post-operation weeks as we previously described^{40 (link)}. Eyeballs were enucleated and fixed in 4% paraformaldehyde for 3 hours. After incubation in 30% glucose solution overnight, retinal tissues were collected and serially frozen-sectioned to a thickness of 10 μm. Immunofluorescence was performed as previously described^{57 (link)}. In detail, sections that crossed the optic disc were rinsed in 0.1 M PBS and blocked for 30 min in 10% of goat serum diluted in 0.1% of Triton X-100. Then, sections were incubated with the primary antibodies, anti-human mitochondrial antibody (1:200, mouse, Abcam), anti-human mitochondrial antibody (1:200, rabbit, Millipore, Billerica, MA), anti-recoverin (1:1000, rabbit, Millipore), anti-rhodopsin (1:8000; rabbit, Sigma-Aldrich), anti-Iba1 (1:500; Wako, Japan) and GFAP (1:500, rabbit, Sigma Chemical Co) in 1% BSA at 4 °C overnight. Secondary antibodies, cy3-or 488-conjugated (Invitrogen), were then implemented (1:400, 37 °C, 2 h). Some sections were processed only with the secondary antibodies as negative controls. Before examination with a confocal laser scanning microscope (Leica, Germany), sections were counterstained with DAPI (Sigma Aldrich, St. Louis, MO, USA).