Peroxidase conjugated anti mouse secondary antibodies

Hippocampus and cortex from the mice with different genotypes (n=15 per group) were harvested and separately homogenized on ice in 10 mM Tris-HCl buffer (pH 7.4), 10 mM EDTA, 30 % Triton-1000, 10 % SDS, protease inhibitors cocktails (Roche), and NaCl, using a homogenizer. Homogenates were centrifuged at 5,000g for 10 min at 4 °C. Equal amounts of protein (quantized by BCA method) were resolved by SDS-PAGE on 4-12% gels, transferred to nitrocellulose, and incubated with antibodies against either Navβ2 (1:500, Alomone) or Nav1.1α (1:800, Alomone). β-actin (mouse monoclonal anti-β-actin, 1:800, Santa Cruz, Delaware, CA) was used as a reference. Nitrocellulose membranes were washed and incubated with peroxidase-conjugated anti-mouse secondary antibodies (1:10,000, Santa Cruz), and were detected using enhanced chemiluminescence reagent (Pierce). Signals were quantified using a ChemiDoc™ XRS+ imaging system with Image Lab™ software (Bio-Rad, USA).

For intracellular HBV surface proteins, cells were lysed with RIPA buffer in the presence of protease inhibitor cocktail (Sigma, United States). For extracellular proteins, supernatants were used directly. Samples were mixed in Laemmli buffer, boiled, loaded on 12% SDS-polyacrylamide gels, and transferred to PVDF membranes (Hybond, GE Healthcare, United Kingdom), according to standard protocols. HBV surface proteins were detected using anti-SHBs (HB01) monoclonal antibody (kindly provided by Prof. Aurelia Zvirbliene, Lithuania). Peroxidase-conjugated anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology (United States). Protein specific bands were visualized using an enhanced chemiluminescence (ECL) system (GE Healthcare, United Kingdom). Quantification was performed using ImageJ software (Wayne Rasband, NIH, United States). β-actin detection was used as intracellular protein loading control.