Rabbit anti bax

The cells were cultivated on glass-bottomed cell culture dishes (Nest Biotechnology, China) at 37 °C for 24 h. Next, cell cultures were subsequently preincubated with either 50 μM BSO or 5 mM OTC for 20 h prior to exposure to DMAE-CB. Then, the cells were treated with 0.01 mM DMAE-CB in the presence or absence of BSO or OTC at 37 °C for 24 h. The cells were then fixed with a modified Zamboni's fixative (4% paraformaldehyde and 0.19% picric acid in PBS, pH 7.4) for 30 min at room temperature. Cells were washed twice with PBS, preincubated in the same buffer containing 0.3% Triton X-100 (Amresco, USA), and blocked with 0.1% BSA (Amresco, USA) in PBS for 1 h. The cells were incubated with primary antibodies (rabbit anti-Bax, 1:200, Beyotime Biotechnology, China; and mouse anti-Cytochrome C, 1:50, Boster Biotechnology, China) overnight at 4°C. After three washes with PBS, cells were incubated with secondary antibodies (Alexa Fluor 594 donkey-rabbit, 1:400; and Alexa Fluor 488 donkey-mouse, 1:200; Life Technologies, Carlsbad, CA) for 30 min at 37°C. Cells were then washed in PBS and further incubated with 10 mg/ml DAPI (Sigma) at room temperature for 10 min. Fluorescent images were obtained by laser scanning confocal microscopy (Keyence Co., Osaka, Japan).

DDP was purchased from hansoh pharmaceutical, Jiangsu. MTT, DMSO, Hoechst 33258 solution, BCA protein kit, PI, AO and ECL system (BeyoECL plus) were purchased from Beyotime Biotechnology, China. Rabbit anti-FSHR antibody was obtained from Abcam, USA. Rabbit anti-Bax, Bcl-2, Caspase-3, Caspase-8, Bad, LC3 and mouse anti-β-Actin were purchased from Beyotime Biotechnology, China. Annexin V-FITC / PI double staining apoptosis kit was from Roche, Germany and Novex NuPAGE Gel Electrophoresis Systems was from Life Technologies, USA. And male BALB/c nude mice were purchased from Cavens Lab Animal, Changzhou, China.