4.7. Confocal Microscopy
HEK293T cells were cotransfected with pMyc-eEF1A (3 μg) and p3×Flag-NS5A (2 μg). After 48-h incubation, the cells were fixed with 4% paraformaldehyde in PBS for 30 min and permeabilized with 0.1% Triton X-100 for 15 min. The cells were then incubated with an anti-Myc PAb (1:100) (catalog no. C3956; Sigma-Aldrich) or anti-Flag MAb (1:100) (catalog no. F1804; Sigma-Aldrich) for 2 h. The cells were incubated with goat anti-mouse IgG (whole molecule)-fluorescein isothiocyanate (FITC) antibody (1:100) (catalog no. F2012; Sigma-Aldrich) and goat anti-rabbit IgG (whole molecule)-tetramethyl rhodamine isocyanate (TRITC) antibody (1:100) (catalog no. T6778; Sigma-Aldrich). Subsequently, the cells were stained with 4,6-diamidino-2-phenylindole (DAPI) (1:1000) (catalog no. D8417; Sigma-Aldrich) for 15 min and examined using a Leica SP2 confocal system (Leica Microsystems, GmbH, Wetzlar, Germany).