Mouse anti chop antibody

The cells were washed once with phosphate-buffered saline (PBS) and then lysed in cold lysis buffer (50 mM TrisHCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, and 1× protease inhibitor cocktail). Cell lysates were centrifuged at 14,000 × g for 15 min at 4°C and then boiled in 5× sample buffer following protein determination (BSA, #23208; Thermo Fisher Scientific, USA). The protein samples were subjected to western blotting analysis. For western blotting analysis, nitrocellulose membranes (#1620145; Bio-Rad, USA), blocking reagent (5% skim milk, 1 h, room temperature), and precasting gels (#456-1095; Bio-Rad) were used with the indicated antibodies at a 1:1,000 dilution ratio. The samples were stained with rabbit anti-EHMT1 antibody (A301-642A; Bethyl Laboratories, USA), mouse anti-CHOP antibody (#2895; Cell Signaling Technology, USA), rabbit anti-PARP antibody (#9542; Cell Signaling Technology) and mouse anti-ACTB antibody (SC-47778; Santa Cruz Biotechnology, USA) at 4°C (overnight). Secondary antibodies (rabbit: SC-2357, mouse: SC-516102; Santa Cruz Biotechnology) were incubated at room temperature for 1 h, and ECL solution (#170-5060; Bio-Rad) was used for visualization.

Eyes were fixed in 4% paraformaldehyde, embedded in optical coherence tomography (OCT) compound (Sakura Finetek), and sectioned using a cryostat (CM3050; Leica Microsystems). The section was blocked with 5% donkey serum for 30 min, incubated with goat anti-Brn3a antibody (sc-31984, 1:100; Santa Cruz Biotechnology), mouse anti-chop antibody (#2895, 1:500; Cell Signaling Technology), or rabbit anti-p53 antibody (sc-6243, 1:100; Santa Cruz Biotechnology) for 1 hr and then stained with an appropriate secondary antibody (anti-goat Alexa Fluor 488, anti-rabbit Alexa Fluor 488, or anti-mouse Alexa Fluor 488; Thermo Fisher Scientific) and DAPI (Vector Labs) for an additional 45 min. For primary antibodies raised in mice, frozen sections were treated in using the Mouse on Mouse Immunodetection kit (BMK-2202, Vector Labs) according to the manufacturer’s instructions. Cells in the GCL were counted in a 1 mm² area on each side of the optic nerve under a 20× objective in five 60-μm-spaced sections from each retina.