Mouse monoclonal anti il 1β

Mouse monoclonal anti-NFκB p65, mouse monoclonal anti-CaMKII, rabbit polyclonal anti-BDNF, and rabbit polyclonal anti-integrin αM antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Mouse monoclonal anti-actin, mouse monoclonal anti-NR2B and rabbit polyclonal anti-NR2A antibodies were purchased from Millipore (Billerica, MA, USA). Mouse monoclonal anti-GFAP, rabbit monoclonal anti-phospho-NFκB p65, rabbit monoclonal anti-TNF-α, rabbit monoclonal anti-IL-6, mouse monoclonal anti-IL-1β, and horseradish peroxidase (HRP)-conjugated IgG antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). ECL Western Blotting Reagents were purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

20 μg of protein extract were resuspended in loading buffer (0.05 M Tris-HCl pH 6.8; 40 g/L sodium dodecyl sulfate; 20 mL/L glycerol; 2 g/L bromophenol blue and 0.02 M dithiothreitol; all from Sigma-Aldrich) and loaded onto 12.5% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE; Bio-Rad Laboratories SrL, Segrate, MI, IT). After electrophoresis and transfer onto nitrocellulose membranes (GE Healthcare Europe GmbH, Milano, MI, IT), membranes were blocked for 1 h in 5% non-fat milk/0.1% Tween-20 in PBS (Sigma-Aldrich), pH 7.4, and incubated overnight at 4°C with primary antibodies (for immunomodulation rabbit polyclonal anti-iNOS from Cell Signalling, rabbit polyclonal anti-TREM2 from Millipore, mouse monoclonal anti-IL-1β from Cell Signalling, rabbit polyclonal anti-BDNF from Santa Cruz Biotechnology and mouse monoclonal anti-GAPDH from Santa Cruz Biotechnology, all 1:1000 except for GAPDH 1:20000) in 0.1% Tween-20/PBS. Membranes were then incubated with an anti-rabbit or anti-mouse secondary antibody conjugated to horseradish peroxidase (1:5000; both from Jackson ImmunoResearch), for 90 min at RT in 0.1% Tween-20/PBS. Labeled proteins were detected by using the enhanced chemiluminescence method (ECL; BioRAD) with a Chemidoc (BioRad) chemiluminescence detector. Densitometric analysis was performed by using Biorad Image Lab software.