Mouse anti human caspase 3 polyclonal antibody

The SW620 cells (6×10⁴ cells/ml) were seeded into six-well plates and treated with 0.25 mg/ml LBF solution for 24 h. The cell monolayer was fixed and treated with 0.5% Triton X-100 (Sigma-Aldrich) for 20 min and 3% H₂O₂ for 15 min. Following blocking with 10% FBS/PBS, primary mouse anti-human caspase 3 polyclonal antibody and rabbit anti-human Bcl-2 polyclonal antibody (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were added and incubated overnight at 4°C, followed by incubation with the goat anti-mouse and goat anti-rabbit, polyclonal, secondary antibody (Santa Cruz Biotechnology, Inc.) at a dilution of 1:200 for 30 min. The sections were visualized by 3-3′-diaminobenzidine (Roche Diagnostics GmbH, Mannheim, Germany) and the untreated cells were used as a negative control.

SMMC 7721 cells treated with 2.8 μg/ml of LMWSVP for 4 h were collected by centrifugation at 1,064 × g for 5 min at 4°C. The cells were then lysed in RIPA buffer, and 50 μg of total protein was separated by 10% SDS-PAGE for 2 h. The separated proteins were transferred to a nitrocellulose membrane (Pall Corporation, Port Washington, NY, USA) using a semi-dry blotter (Bio-Rad) for 1 h and then blocked with 5% non-fat milk for 1 h. The specific primary antibodies, at an optimized dilution, were incubated with the membrane [mouse anti-human Caspase-3 polyclonal antibody 1:1,000; rabbit anti-human Bcl-2 polyclonal antibody 1:1,000; mouse anti-human β-actin polyclonal antibody 1:10,000 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA)] and incubated overnight at 4°C. Following incubation with secondary horseradish peroxidase-conjugated antibody (goat anti-mouse and goat anti-rabbit,1:10,000) for 1 h, the protein bands were visualized by enhanced chemiluminescence (Santa Cruz Biotechnology, Inc.).