Horseradish peroxidase labeled anti mouse antibody

Proteins obtained from peritoneal macrophages were extracted by scraping and collected in ice-cold PBS supplemented with a mixture of protease and phosphatase inhibitors. The method described by Bradford et al. was employed to quantify the protein concentration. 45 Samples with equal amounts of protein (15 µg) were separated by 10% or 15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane, followed by blocking it with a blocking solution buffer for 1-2 h at room temperature and incubation overnight at 4 °C with specific primary antibodies (Table 1). After rinsing, the membranes were incubated with a horseradish-peroxidase-labeled anti-mouse antibody (Dako, Atlanta, GA, USA) or an anti-rabbit antibody (Cell Signaling Technology, Danvers, MA, USA) containing blocking solution for 1-2 h at room temperature. The blots were analyzed for β-actin expression in order to prove equal loading. An enhanced chemiluminescence light detection kit was used for immunodetection (Pierce, Rockford, IL, USA). Immune signals were captured using the Amersham Imager 600 from GE