Horseradish peroxidase hrp conjugated goat anti rabbit igg secondary antibody

Total protein was extracted from 2.0 mm tissue punches of total MPOA and NAc. Tissue samples were homogenized in extraction buffer, and supernatant was extracted according to methods of Lee (2007) . Protein estimates were calculated from this supernatant using spectrophotometry (PowerWaveX; BioTek, MD). These protein estimates were used as a standard for loading volume (35 μg of protein each) onto 12% polyacrylamide gels for separation by SDS electrophoresis (Lee, 2007 ). Western immunoblots were processed as previously described (see McHenry et al., 2012 (link)). The membranes were incubated overnight at 4 °C in blocking solution and FosB rabbit polyclonal antibody (1:1k, 5G4 Mab #2251, Cell Signaling, MA) and the next day for 90 min in horseradish peroxidase (HRP)-conjugated goat antirabbit IgG secondary antibody (1:500, SC-2030, Santa Cruz Biotechnology, CA). The membrane was assessed for HRP-conjugated chemiluminescence (ECL; Amersham Biosciences, NJ) by exposing the membrane to Kodak BioMax film. The membrane was then stripped and labeled against mouse anti-β-actin monoclonal antibody (1:1k, Sigma-Aldrich, MO) as a loading control. The developed films were scanned into a computer, and band density was quantified using the NIH ImageJ program (Version 1.33U, NIH).