Peroxidase conjugated goat anti mouse antibody

A western blot analysis was performed as described previously [18 (link)]. Cell lysates were resolved by SDS-PAGE and transferred to a low-fluorescence PVDF membrane (Azure Biosystems, Dublin, CA, USA). Individual proteins were labelled with specific antibodies and visualized by chemiluminescence and SuperSignal^® West Femto Maximum Sensitivity Substrate (Thermo Scientific, Waltham, MA, USA) or by near infrared fluorescence using Azure Biosystems c600. IRF-3 was detected with a mouse monoclonal antibody (BioLegend, San Diego, CA, USA, dilution 1:1,000) and a peroxidase-conjugated goat anti-mouse antibody (Sigma Co., St. Louis, MO, USA, dilution 1:10,000); phosphorylated IRF-3 was detected with a rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) and a IR 700-conjugated goat anti-rabbit antibody (Azure Biosystems, Dublin, CA, USA, dilution 1:10,000); β-actin was detected with a rabbit polyclonal antibody (Abcam, Cambridge, UK, dilution 1:2,500) and peroxidase-conjugated goat anti-rabbit antibody (MP Biomedicals-Cappel, Solon, OH, USA, dilution 1:10,000); PARP-1 was detected with a rabbit polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA, dilution 1:500) and peroxidase-conjugated goat anti-rabbit antibody (MP Biomedicals-Cappel, Solon, OH, USA, dilution 1:10,000).

Recombinant human TF purified from SF9 cells (Haematologic Technologies Inc, Essex, VT, USA) was adsorbed to microtitration plates by overnight incubation of protein diluted 10 µg/ml in PBS (phosphate buffered saline) pH 7.4 at 4°C. After block with BSA (bovine serum albumin) and washing, scalar dilutions of the tested antibody (from 1000 ng/ml to 10 ng/ml) were incubated 1 hour at room temperature, and then detected by a peroxidase-conjugated goat anti-mouse antibody (Sigma-Aldrich, St Louis, MO, USA).