Immunoblotting Analysis of Sodium Channels

To obtain total lysates for immunoblotting analyses, primary hippocampal neurons were washed in phosphate buffered saline (PBS) and collected by gentle scraping in ice-cold RIPA buffer containing (in mM) 50 Tris pH 7.4, 100 NaCl, 1 EGTA, 1 PMSF, 1 sodium orthovanadate, 1 NaF, 0.5% NP-40, and 0.2% SDS supplemented with protease inhibitor cocktail II (Roche Diagnostic, Monza, Italy). Nitrocellulose membranes were incubated overnight with rabbit-polyclonal anti-Na_V1.6 (1:500, Alomone Labs-Israel)^{22 (link),80 (link)}, anti-Na_V1.2 (1:500, Alomone Labs-Israel)^{22 (link)} and anti-Na_V1.1 antibodies (1:500, Alomone Labs-Israel)^{65 (link)}, or mouse monoclonal anti-tubulin (1:3000, Sigma Aldrich, Milan, Italy). Immunoreactive bands were detected with the chemiluminescence system (Amersham-Pharmacia-Biosciences, UK). Films were developed with a standard photographic procedure and the quantitative analysis of detected bands was carried out by densitometric scanning.