P creb1

Retinal slides were fixed with 4% paraformaldehyde at room temperature for 20 min and subsequently incubated with 0.5% Triton X-100 for 10 min. Then, the samples were treated with a blocking solution (5% normal goat serum and 2% bovine serum albumin in PBS) for 30 min to prevent nonspecific antibody–antigen binding. γ-H2AX, ligase IV, Nrf-1, CREB1 and P-CREB1 expression was detected using the antibody of γ-H2AX (Millipore), ligase IV (Santa Cruz), CREB1 (CST), P-CREB1 (CST) and Nrf-1 (Santa Cruz). For the fluorescence visualization of antibody reactions, the primary antibodies were detected using secondary antibodies labeled with the fluorochromes Alexa Fluor 555 or 488 (CST), while the nuclei were detected with DAPI.

Briefly, PBS was used to wash the slides three times, which were fixed in 4% paraformaldehyde. Ten minutes later, 0.1% Triton X-100 in PBS was employed to permeabilize cells for 15 min. Then, tissues were blocked with 2% BSA in TBST for 1h. The antibody (NeuN [1:400, cat. No. ABN78; Millipore], p-CREB1 [1:200, cat. No. ab32096; Abcam], β3-Tubulin [1:400, cat. No. MAB1637; Millipore], and caspase-3 [1:100, cat. No. sc-7272; Santa Cruz]) was incubated overnight, and the second antibody was incubated for 1h. DAPI was used to stain the nuclei. Subsequently, the images were photographed using OLYMPUS FV1000.