Alexa fluor 488 conjugated goat anti mouse immunoglobulin igg

BHK‐21 cells and C6/36 cells were cultured in 24‐well plates containing a coverslip inside at a density of 5 × 10⁴ cells/500 μl per well overnight. The cells were infected with viruses at a MOI of 10 for 2 hours. The plates were maintained at 37°C with 5% CO₂. At 48 hours post‐transfection, the cells were washed with PBS. The coverslips were fixed in 4% paraformaldehyde for 15 minutes and then washed with PBS. The cells were permeabilized with 0.5% Triton X‐100/PBS at room temperature for 20 minutes. The coverslips were subsequently blocked with 5% bovine serum albumin. Each coverslip was incubated with 300 μl of 1:100 diluted DENV monoclonal antibody and placed in a wet box at 4°C overnight. Samples were incubated with a 500‐fold dilution of Alexa Fluor 488 conjugated goat anti‐mouse immunoglobulin (Ig)G (Invitrogen, Carlsbad, CA, USA) in a wet box, avoiding light at 37°C for 45 minutes. The coverslips were fixed to glass slides and the cell nuclei were stained with 4',6‐diamidino‐2‐phenylindole (Sigma) and anti‐fluorescence quencher. Images were observed and collected using an upright fluorescence microscope.