Ready set go mouse tnf α kit

Primary cortical neuron-BV2 co-cultures were fixed by incubating the cells in 4% PFA for 20 min at RT. Subsequently, cells were permeabilized in methanol containing 0.3% H₂O₂ for 10 min. Prior to MAP2 staining, nonspecific binding was inhibited by incubation with PBS containing 1% BSA and 10% horse serum (Vector Labs, Orton Southgate, Peterborough, UK) for 20 min, at RT. Neuronal cells were immunolabeled with anti-MAP2 antibody (1:2000, Sigma-Aldrich, St. Louis, MO, USA, M9942) overnight at 4 °C. Next, cells were incubated with biotinylated horse anti-mouse secondary antibody (1:200, Vector Labs, Orton Southgate, Peterborough, UK) for 1 h. ExtrAvidin-HRP tertiary antibody staining (1:500, Sigma-Aldrich, St. Louis, MO, USA) was performed for 1 h, at RT. To develop the staining, cells were incubated with the ABTS peroxidase substrate (Vector Labs, Orton Southgate, Peterborough, UK) for 30 min. From each well, 150 μL of substrate solution was collected and absorbances was measured at 405 nm with a microplate reader (BioRad, Hercules, CA, USA). Levels of secreted TNF-α were determined from 1:2 diluted conditioned culture media with a Ready-SET-Go mouse TNF-α kit (eBioscience, San Diego, CA, USA).