Anti rabbit igg h l hrp conjugate

The protein levels of VEGF and caspase-3 p17 in bone tissue were determined by western blot analysis. Bone tissue samples were lysed in RIPA buffer containing protease inhibitor cocktail (PIC, Sigma, USA). Lysate samples (50 µg protein) were electrophoresed on 15% SDS-PAGE gel and then transferred onto nitrocellulose membranes. The membranes were blocked and then incubated with anti-VEGF antibody (1 : 500, Santa Cruz Biotechnology, USA) and anti-caspase-3 antibody (1 : 1000, Santa Cruz Biotechnology, USA) overnight at +4°C followed by incubation with secondary antibodies: anti-rabbit IgG (H + L)-HRP conjugate (1 : 4000; Bio-Rad Laboratories, Inc., USA) and anti-mouse IgG (Fab specific)-Peroxidase (1 : 2500; Sigma, USA) for 1 h at room temperature. Thereafter, the membranes were developed with chemiluminescent agents: p-coumaric acid (Sigma, USA) and luminol (AppliChem GmbH, Germany). VEGF and caspase-3 tissue levels were normalized to β-actin (1 : 10000; Sigma, USA). The immunoreactive bands were quantified with Gel-Pro Analyzer v3.1 software.