Fluoview fv10 asw 4.1 software package

Monolayers of BeWo and HUVEC cells were infected with 100 infectious units per well. After 48 h, samples were fixed with 4% paraformaldehyde and blocked with 5% lamb serum. Cells were stained with anti-CHIKV monoclonal antibody 3E7b and anti-MAP2 antibody (Novus Biologicals, Littleton, CO, USA). Slides were mounted with ProLong Gold Antifade Reagent with DAPI (Cell Signaling Technology, Danvers, MA, USA catalog #8961S) and images were obtained using an Olympus Fluoview 3000 confocal microscope. Images were processed using the Olympus Fluoview FV10-ASW 4.1 software package.

Prior to proceeding with the co-culture assay, we verified the permissiveness of BeWo and HUVEC cells to our ZIKV isolate. Confluent BeWo and HUVEC monolayers were infected with 1000 plaque-forming units per well in a 12-well plate. After 48 h, the samples were fixed with paraformaldehyde solution, 4%, in phosphate buffered saline (PBS) (ThermoScientific CAT# J19943-K2) and blocked in 5% lamb serum. Primary antibody staining with an anti-flavivirus-group antigen, Clone D1-4G2-4-15 (BEIresources NR-50327), was performed to visualize ZIKV. The staining was conducted overnight at 4 °C. The cells were rinsed in PBS and then incubated with secondary antibodies at room temperature for 1 h. The secondary antibodies included AlexaFluor 647 goat anti-mouse (Invitrogen #A21235). The cells were rinsed, and then, coverslips were mounted with ProLong Gold Antifade Reagent with DAPI (Cell Signaling Technology, Danvers, MA, USA, catalog #8961S) and incubated overnight at 4 °C. Images were taken with an Olympus Fluoview 3000 confocal microscope and processed using the Olympus Fluoview FV10-ASW 4.1 software package.