2.9. NF-κB Nuclear Localization for Immunofluorescence Staining

NF-κB (p65) nuclear localization was determined by immunofluorescence assays using a fluorescence microscope. SK-N-SH cells were cultured directly on glass coverslips in four-well plates for 24 h. After stimulation with HSV-1 strains (MOI = 0.01) in the presence or absence of QA, the cells were fixed with 4% paraformaldehyde in PBS. The membrane was permeabilized by treating the cells for 5 min with 0.1% Triton X-100 in PBS. After a brief washing in PBS, the slides were blocked with 5% bovine serum albumin for 1 h and then incubated with rabbit polyclonal anti-human phopho-p65 antibody (dilution, 1:100) for 1 h at room temperature. After washing, the cells were incubated with the secondary antibodies (Alexa Flour 488, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min, and the nuclei were counterstained with Hoechst 33342 (ImmunoChemistry, Bloomington, MN, USA) for 10 min. The slides were mounted using ProLong^® Gold antifade reagent (Molecular Probes^® by Life Technologies, Carlsbad, CA, USA). Fluorescent micrographs were acquired with a fluorescence microscope (Nikon ECLIPSE Ti-U, Nikon Co., Tokyo, Japan).