Rabbit anti caspase 3

Five-micrometer tissue sections were deparaffinized in xylene, rehydrated in an ethanol series, and submitted to antigen retrieval step. The sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) was used for heat-induced epitope retrieval. After 15 min of antigen retrieval step, the sections were blocked for nonspecific binding with 5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature and incubated with the primary antibodies for 18 h at 4°C. These sections were washed with PBS three times and then were incubated with primary antibodies, including rabbit anti-Caspase 3 (1:100; Chemicon), rabbit anti-PARP (1:100, Promega), and rabbit anti-NF-κB (1:100; R&D Systems, Minneapolis, MN, USA).

The total protein was isolated from liver tissues using RIPA buffer (Sigma-Aldrich, St. Louis, US). Ten micrograms of total protein was loaded per well and then separated on 12% sodium dodecyl sulfate-polyacrylamide gel. Then, the protein was transferred onto the polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, United Kingdom). The membrane was blocked with 5% BSA in TBST (0.5% Tween 20) for 1 h at room temperature. The membrane was incubated at 4°C overnight with primary antibodies: rabbit anti-phospho-AKT (Ser473, 1 : 1000), rabbit anti-phospho-Stat3 (Tyr705, 1 : 2000), rabbit anti-phospho-NF-κB p65 (Ser536, 1 : 1000), rabbit anti-AKT (1 : 1000), rabbit anti-Stat3 (1 : 2000), anti-NF-κB p65 (1 : 1000), rabbit anti-caspase 3 (1 : 1000), and rabbit anti-GAPDH (1 : 20000, Sigma-Aldrich, St. Louis, USA). All antibodies besides rabbit anti-GAPDH were purchased from Cell Signaling Technology, Beverly, USA. The membrane was incubated for 1 h at room temperature with a goat polyclonal to rabbit IgG antibody (1 : 10000, Abcam) and developed with the SuperSignal West Femto Trial kit (Thermo, USA). The membrane was exposed to high-sensitivity films (GE Healthcare) for autoradiography.

Western blots were performed as described [34 (link)]. Briefly, proteins were separated by gel electrophoresis, transferred to the Hybond-P PVDF membrane, blocked with 2% milk in PBST, incubated with primary and HRP secondary antibodies, treated with luminol substrate and developed on a film. Mouse monoclonal anti-caspase 8 (Sigma-Aldrich), mouse monoclonal anti-caspase 9 (Sigma-Aldrich), rabbit anti-caspase 3 (Sigma-Aldrich), rat monoclonal anti-caspase 12 (Sigma-Aldrich) and rabbit polyclonal antibody against Bim (amino acids 4–195 of Bim_EL form) were all used at a 1:1000 dilution for Western blots.

Western blots were performed as described [38] (link). Mouse monoclonal anti-Caspase 8 (Sigma), mouse monoclonal anti-Caspase 9 (Sigma), rabbit anti-Caspase 3 (Sigma), rat monoclonal anti-Caspase 12 (Sigma), rabbit polyclonal antibody against Bim (amino acids 4–195 of Bim_EL form), rabbit anti-Cytochrome C (Cell Signaling), goat polyconal anti-HP1 (Santa Cruz), rabbit anti-CoxIV (Cell Signaling), rabbit anti-phospho-JNK (Thr183/Tyr185) (81E11) (Cell Signaling), rabbit anti-JNK (Cell Signaling), mouse monoclonal anti-CHOP (L63F7) (Cell Signaling), rabbit anti-Apaf-1 (Cell Signaling), rabbit anti-Bcl2 (Cell Signaling), rabbit polyclonal anti-ATF-6 (Santa Cruz), rabbit anti-Bax (Cell Signaling) were all used at a 1:1000 dilution for western blots.