Cleaved caspase 3 monoclonal antibody

Staurosporine was purchased from MilliporeSigma (Billerica, MA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), tert-butyl hydrogen peroxide (t-BHP), N-acetylcysteine (NAC), dimethyl sulfoxide (DMSO), and metaphosphoric acid were all purchased from Sigma-Aldrich (St. Louis, MA). Cleaved caspase-3 monoclonal antibody, anti-rabbit IgG horseradish peroxidase (HRP)-linked antibody, and SignalFire Enhanced Chemiluminescent Reagents were purchased from Cell Signaling Technology (Danvers, MA). The GSH/GSSG Assay Kit was purchased from Aoxre Biosciences-Oxis Research (Portland, OR).

Protein levels of cleaved caspase-3 in rat livers were determined by Western blot analysis. Proteins extracted from livers were subjected to SDS-PAGE gels and electrotransferred to polyvinylidene fluoride (PVDF) membranes. Nonspecific binding sites were blocked in 5% bovine serum albumin at room temperature for 1 h. Membranes were subsequently probed with primary antibodies at 4 °C overnight, then incubated with the appropriate peroxidase-conjugated secondary antibody. Cleaved caspase-3 monoclonal antibody (1:1000; Cell Signaling Technology, Boston, MA, USA) and Cyp7a1 antibody (1:1000; Shenggong BBI Life, Shanghai, China) were used as primary antibodies, and horseradish-peroxidase-conjugated anti-rabbit IgG (1:5000; Abclonal, Wuhan, China) was used as a secondary antibody. Densitometry of immunoblot analysis was performed using ImageJ software as previously described [14 (link)].

For cleaved caspase-3 staining, bone sections after the blocking step were incubated with a monoclonal cleaved caspase-3 antibody (Cell signaling Technology Inc., Danvers, MA, USA) (dilution 1:200) and then incubated with a secondary antibody Dylight^TM 488 anti-rabbit (Rockland Immunochemical, Pottstown, PA, USA) (dilution 1:400). Bone sections were washed and then stained with DAPI for nucleus detection and mounted in Vectashield^TM mounting media (Vector Laboratories, Burlingame, CA, USA).