4.4. Western Blot Analysis

Mouse brain tissue and primary neuronal cells were lysed in a buffer containing: Tris-HCl (20 mM, pH 7.5); NaF 10 mM; NaCl 150 mM; phenylmethylsulphonyl fluoride (PMSF) 1 mM; NONIDET P-40 1%; Na3VO4 1 mM; aprotinin 0.1%; pepstatin 0.7 mg/mL e leupeptin 1 μg/mL. Homogenates were centrifuged at 14,000 rpm for 20 min at 4 °C. The supernatant was used to perform Western blot analysis. Protein levels were determined using the Bradford method. The total protein amount used for each sample was 50 μg and it was separated on 8% or 15% sodium dodecyl sulfate-polyacrylamide gels with 5% sodium dodecyl sulphate stacking gel (SDS-PAGE) and electrotransferred onto Hybond ECL nitrocellulose paper (Amersham, Milan, Italy). The membranes were blocked in 5% non-fat dry milk in 0.1% Tween 20 (TBS-T; 2 mmol/L Tris HCl, 50 mmol/L NaCl, pH 7.5) for 1 h at room temperature (RT) and subsequently incubated overnight at 4 °C in the blocked buffer with the 1:1000 antibody for NCX1 (polyclonal rabbit antibody, Swant, Marly, Switzerland), 1:5000 antibody for NCX3 (polyclonal rabbit antibody, Philipson’s Laboratory, UCLA, Los Angeles, CA, USA), 1:1000 GFAP (polyclonal, Novus Biologicals, Littleton, CO, USA; NB300-141), 1:1000 for IBA-1 (polyclonal, Wako 019-19741), 1:1000 for nNOS (monoclonal, Santa Cruz, Dallas, TX, USA; NOS1(R-20)sc-648), 1:1000 for iNOS (monoclonal, NOS2, Santa CRUZ (C-11):sc-7271), 1:1000 for IL-1β (monoclonal, Cell Signaling, Danvers, MA, USA; 12242), 1:1000 for Cyt c (polyclonal, Cell Signaling 4272). Next, all membranes were washed 3 times with a solution containing Tween 20 (0.1%) and subsequently incubated with the secondary antibodies for 1 h (1:2000) at room temperature. Immunoreactive bands were detected by ECL (Amersham). Discrimination among the distinct types of extracts was ensured by running parallel Western blots with the endogenous β-actin or α-tubulin protein. The optical density of the bands was determined by the Image J program.