Mouse monoclonal anti inos

Proteins were extracted from brain tissues (100 mg), BV-2 cells (1 × 10⁵), and N2a cells (1 × 10⁵), respectively, and were subjected to SDS-PAGE. Separated proteins were transferred onto nitrocellulose membranes (Millipore Corp, Billerica, MA) and were blocked with 5% skimmed milk. After blocking, the membranes were incubated with the primary antibodies, including mouse monoclonal anti-NeuN (1:1 000, Chemicon, Temecula, CA), mouse monoclonal anti-iNOS (1:500, BD, Bio-science), and mouse monoclonal anti-actin (1:500, Santa Cruz, CA). After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10 000, Zhongshan BioTech Co, China). The protein bands were visualized by using an ECL kit (Super Signal West Pico, Thermo Scientific, USA).

Ceruloplasmin from human plasma was purchased from Enzo Life Sciences (Farmingdale, NY, USA), the presence of endotoxin contaminant was evaluated using the Limulus Amebocyte Lysate (LAL) Pyrogent® Plus Single test (Lonza, Walkersville, MD, USA) and results found to be 0.006 EU/μg of Cp. E. coli LPS, N_ω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and other chemicals, when not specified, were obtained from Sigma-Aldrich (St Louis, MO, USA). Recombinant rat IL-1β, TNF-α, INF-γ and granulocyte macrophage colony-stimulating factor (GM-CSF) were from R&D Systems (Minneapolis, MN, USA). All the reagents were resuspended in apyrogenic endotoxin-free water for clinical injectable preparations (SALF-Laboratorio Farmacologico, Bergamo, Italy). The antibodies used in the study were mouse monoclonal anti-iNOS (BD Biosciences, San Jose, CA, USA), mouse monoclonal anti-α-tubulin (Sigma-Aldrich, St Louis, MO, USA). Polyclonal goat anti-mouse Ig horseradish peroxidase (HRP)-conjugated (DAKO, Carpinteria, CA, USA) was used as the secondary antibody.