Superblock t20

Basically, the analysis was performed using methods previously reported by our group^{23 (link)}. Proteins were extracted from samples using NP-40 Cell Lysis Buffer (Thermo Fisher Scientific, MA, USA) supplemented with Complete protease inhibitor (Sigma Aldrich) and Halt Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). Equal amounts of protein were separated by SDS-PAGE (12.5%), transferred onto a nitrocellulose membrane, and blocked with SuperBlock T-20 (PBS; ThermoFisher Scientific). Primary antibody of cleaved caspase-3 (Cell Signaling Technology, Inc, MA, USA, #9661) were diluted to 1:1000 in SuperBlock T-20 (PBS) and incubated with blots overnight at 4 ℃. After washing in T-PBS, blots were probed with HRP-conjugated secondary antibodies (R&D Systems, Inc., MN, USA), washed in T-PBS, and then developed using the Super-Signal West Pico enhanced chemiluminescence system (ThermoFisher Scientific). Protein expression levels were normalized to the actin expression (BD Transduction Laboratories, KY, USA).

After 12 weeks of treatment, rat retinas were harvested and washed with cold PBS, followed by incubation in RIPA buffer (Solarbio, China) on ice. Retinas were extracted and protein content was quantified using a BCA assay kit (Cwbiotech, China). After loading buffer was added, cell lysates were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Membranes were blocked using Super Block T20 (TBS) buffer and incubated with primary antibodies (Cell Signaling Technology, USA) overnight at 4 °C, then washed 3 times with TBST [tris-buffered saline (TBS) and Tween 20]. Dilution ratios: mouse monoclonal antibodies against ICAM-1 (1:1000), rabbit monoclonal antibodies against TNF-α (1:1000), rabbit polyclonal antibodies against NF-κB (1:1000), rabbit polyclonal antibodies against p38-MAPK (1:1000). The membrane was then incubated with HRP-conjugated secondary antibodies (Jackson Immuno Research, USA) for 1 h, at the following dilution ratios: goat anti-rabbit, 1: 5000; goat anti-mouse, 1: 5000. Enhanced chemiluminescence (Millipore, USA) was used to visualize the bands and an image analyzer (Bio-Rad, USA) was employed for densitometric analysis. Protein expression was normalized to that of GAPDH (Abcam, USA).