Polyclonal rabbit anti opa1

Immunohistochemical staining of 7-um frozen sections of full-thickness retina was prepared. Three sections per frozen block from each group (n = 3 retinas/group) were used for immunohistochemical analysis. The RGCs (n = 3 per group) on the coverslips were fixed with 4% PFA in PBS for 20 min, rinsed with PBS, and then were used for immunohistochemical analysis. The samples were permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature, sequentially, blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature, incubated by 16 h at 4°C with the following primary antibodies: monoclonal mouse anti-GFAP antibody (1:200; Abcam), polyclonal rabbit anti-OPA1 (1:200; Abcam), and monoclonal rabbit anti-LC3 (1:1000; Abcam) for 16 h at 4°C. After several washes, the samples were incubated with Alexa Fluor 488-conjugated goat IgG secondary antibody (1:200; Life Technologies) and Hoechst 33342 (1 μg/mL; Life Technologies). Fluorescence imaging and analyses were performed by a confocal microscopy (Leica SP8).

Retinas (n = 3 per group) were lysed in RIPA buffer (Beyotime, China) and ultrasonically smashed on ice to get homogenized solutions. RGCs (n = 3 per group) were mixed with RIPA buffer. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the electrotransferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, United States). After blocking with 5% non-fat dry milk at room temperature for 1 h, the membranes were incubated with polyclonal rabbit anti-OPA1 (1:1000; Abcam), polyclonal rabbit anti-GFAP antibody (1:10000; Abcam), polyclonal rabbit anti-Parkin (1:1000; Abcam), polyclonal rabbit anti-Bcl-2 (1:500; Abcam), monoclonal rabbit anti-Bax (1:1,000; Abcam), monoclonal rabbit anti-LC3 (1:2000; Abcam), polyclonal rabbit anti-LAMP1 (1:1000; Abcam) and polyclonal rabbit anti-GAPDH (1:2000; Yesen, China) in primary antibody dilution (Beyotime, China) overnight at 4°C. After the membranes were washed several times, secondary antibody peroxidase-conjugated goat anti-rabbit IgG (1:5000; Jackson) was applied. Proteins were visualized by a Kodak Image Station 4000MM PRO (Carestream, Rochester, NY, United States) using chemiluminescence detection (SuperSignal West Femto Substrate Trial Kit, Thermo Fisher). Band intensity was analyzed with Image J (National Institutes of Health).