Ripa buffer

Total protein was extracted from cells or retina tissues utilizing RIPA buffer (Solarbio, Beijing, China). RIPA buffer (Solarbio, Beijing, China) was used to lyse cells or retina tissues. The protein concentration was determined using the BCA protein assay kit (Pierce, Appleton, WI, USA). Then, protein was separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, followed by transference onto polyvinylidene difluoride (PVDF; EMD Millipore, Burlington, MA, USA) membranes. The membranes were blocked with 5% skim milk for 1 h. Then, the membranes were incubated with primary antibodies including anti-p53 (1:500, ab131442; Abcam, Waltham, MA, USA), anti-Bcl-2 (1:1,000, ab182858; Abcam, Waltham, MA, USA), anti-Bax (1:1,000, ab32503; Abcam, Waltham, MA, USA), anti-Cleaved-Caspase-3 (1:500, 33199M; BSM, Shanghai, China) and anti-β-actin (1:2,000, 20536-1-AP; Proteintech, Wuhan, China) overnight at 4 °C, followed by secondary antibodies (1:6,000, ZB-5301; ZSGB-BIO, Beijing, China) for 30 min at room temperature. The proteins were visualized using an ECL kit (KeyGen Biotech Co., Ltd., Jiangsu, China). β-actin was used as an internal control. The expression of protein was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

After thawing, the tissues were washed twice with deionized water to remove blood contamination on the surface. Then, samples were minced with scissors on ice and homogenized in RIPA buffer (Solarbio, R0010) containing 1% phenylmethylsulfonyl fluoride (PMSF) at a concentration of 10 μL/mg. Then, the homogenates were lysed at 4 ℃ for 30 min and centrifuged at 12,000g for 30 min at 4 ℃. For cell protein extraction, the cells were lysed using 100 μL of RIPA buffer (Solarbio, R0010) containing 1% PMSF after treatment for 24 h in 6-well plates and then centrifuged at 12,000g for 30 min at 4 ℃. Supernatants were collected as protein samples and stored at −80 ℃ until use.