Nis elements imaging system software

Vero cells seeded onto coverslips were inoculated with 150 µL of 9 dpi culture fluid derived from ZIKV infected nonhuman primate tissues and incubated at 37°C for 2 hrs with gentle rocking. Subsequently, media was replaced and infected cells were incubated for 22 hrs to allow virus growth, and then fixed for 30 min with 4% (w/v) paraformaldehyde in TBS. Fixed cells were washed with TBS and permeabilized for 10 min at −20°C with methanol. Cells were blocked with 5% (v/v) normal goat serum in TBS (NGS/TBS), and were stained with ZV-13 mouse monoclonal antibody reactive to ZIKV E protein (provided by Dr. M. Diamond, Washington University) in NGS/TBS.^{50 (link)} Cells were subsequently incubated with goat anti-mouse IgG-Alexafluor 488 conjugate (cat# A-11029, Thermo Fisher Scientific, MA) in NGS/TBS, nuclei were counterstained with DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride, cat# D1306, Thermo Fisher Scientific), and mounted on slides for analysis. Confocal immunofluorescence images were acquired using a Nikon Eclipse Ti microscope and analyzed using NIS-Elements imaging system software (version 4.51) (Nikon Instruments).