Goat anti mouse rabbit secondary antibody

Western blotting was performed as previously described [25 ]. Primary astrocytes were harvested for protein extraction after the different treatments. Mice were anesthetized at different time points after ICH induction, and the brain tissue around the bleeding site was used for WB. The following antibodies were used: rabbit anti-Homer1 (1:1000; Abcam), rabbit anti-C3 (1:1000; Abcam), rabbit anti-S100A10 (1:1000; Proteintech), mouse anti-GAPDH (1:10,000; Abcam), rabbit anti-Ras (1:1000; CST), rabbit anti-Phospho-c-Raf (Ser338) (1:1000; CST), mouse anti-Raf-1 (1:1000; Proteintech), rabbit anti-Phospho-MEK1/2 (Ser217/221) (1:1000; CST), mouse anti-MEK1/2 (1:1000; CST), rabbit anti-Phospho-ERK1/2 (Thr202/Tyr204) (1:1000; Proteintech), rabbit anti-ERK1/2 (1:1000; Proteintech), and goat anti-mouse/rabbit secondary antibody (1:10,000; Abcam).

Total proteins from cells were extracted using RIPA lysis buffer (Beyotime Biotechnology, China) containing protease inhibitor (Roche, Switzerland). Protein samples were subjected to SDS-PAGE and then transferred onto PVDF membranes (Roche, Switzerland). After being blocked with 5% nonfat milk for 2 h at room temperature, the membranes were incubated with primary antibodies (Abcam, UK; 1:1000 dilution) against AKR1B10, Ki67, PCNA, Bcl-2, Bax, cleaved-caspase3 (c-caspase3), pro-caspase3, c-caspase9, pro-caspase9, p53, p21, HOXA5 and GAPDH at 4°C overnight. The membranes were then being incubated with a goat anti-mouse/rabbit secondary antibody (Abcam, UK) for 2 h at room temperature. Enhanced chemiluminescence reagent (Thermo Fisher Scientific, Inc., USA) was used to visualize the protein bands in a Bio-Rad ChemiDoc XRS Imaging system (Hercules, USA).