Hoechst dye

The cells were cultured in CMEM containing 10 µM of 5′-azacytidine (5′-Aza) to stimulate cardiomyogenic differentiation, and the expression of structural cardiac proteins was detected using the cardiac Troponin T (cTNT) (Abcam) antibody. Untreated and 5′-aza-treated cells were grown at a density of 5 × 10⁴ cells/well in 12-well plates (BD Falcon). Cells were rinsed in 1X PBS after 24 h of incubation and then cultured in CMEM alone for 21 days. On day 21, the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) and permeabilized for 15 min with 0.5% Triton X-100 in PBS. Blocking was done with 5% goat serum to minimize non-specific binding. The cells were blocked and incubated overnight at 4 °C with the following antibodies: cTNT at a dilution of 1:200. The cells were then washed thrice and incubated with 1:1000 diluted FITC-labeled anti-rabbit secondary antibodies for 1 h at room temperature in the dark. Cells were rinsed thrice with 1X PBS and counterstained with 500 µL of Hoechst dye (Sigma-Aldrich). The acquisition of the stained images was done by a confocal microscope (Zeiss, Peabody, MA, USA), and their analysis was performed by its software, ZEN-blue software (version 2.3, Zeiss Microscopy GmbH, Germany).

Fluorescence microscopy studies on the cell area were performed using a Nikon Eclipse TE2000-S (Nikon Instruments, Japan) inverse fluorescence microscope with a LH-M100C mercury lamp and GFP (G) bandpass filter (exc. 480, em. 535 nm). A 20x air objective and a 10x were used. Staining of the cell body was done with Calcein AM (1:100) (Sigma Aldrich, USA). Staining of the nucleus was performed using Hoechst dye (Sigma Aldrich, USA). Micrographs were processed with the Fiji distribution of ImageJ and the ZEN Imaging Software (Zeiss). The cell area of at least three independent samples was measured for all cells in the area of the micrograph at 20 °C.

For immunofluorescence analysis of HCV infectivity and TCID₅₀ [21 (link)], the wells were washed and fixed with ice-cold methanol before blocking with 3% of BSA. Samples were then treated at 37°C for 1 h with the mouse monoclonal anti-NS5A 9E10 antibody (1:25,000; gift from Dr. Charles M. Rice), followed by PBS washes three times before detection using the Alexa Fluor 488 goat anti-mouse IgG (H + L) antibody (Invitrogen; 1:400). Following incubation at 37°C for 1 h, the samples were washed with PBS three times prior to staining the nuclei with Hoechst dye (Sigma) and visualization by fluorescence microscopy. Micrographs were taken from 3 random fields per sample for each independent experiment.

Cells fixed with 4% paraformaldehyde, and permeabilized at room temperature in 0.1% Triton X-100 for 30 minutes. After blocking with blocking buffer (Roche), the cells were incubated with primary antibody overnight at 4°C and with secondary antibody for 2 hours at room temperature. The primary antibodies were used at the following dilutions: anti-ST6Gal1 (ST6Gal1-M2, IBL, 1:50), SNA (Vector Laboratories, 1:100), MALII (Vector Laboratories, 1:100), and ABCG2 (R&D, 1:50). Cells were counter-stained with Hoechst dye (Sigma-Aldrich) to visualize the cell nuclei. Immunostaining images were obtained using a fluorescence microscope (Leica Microsystems Inc).