Immunofluorescence Assay for ZIKV Infection

Infected cells from the replication growth kinetics assay were fixed with 4% PFA at days 4, 7, 11 and 17 post infection, permeabilized with 70% ethanol and stained using an immunofluorescence assay (IFA) as described previously^{25 (link)}. Briefly, cells were incubated with mouse monoclonal antibody anti-flavivirus group antigen (MAB10216) clone D1–4G2-4-15 (Millipore, Germany) followed by staining with goat anti-mouse IgG conjugated with Alexa Fluor 488 (Life technologies, the Netherlands). After incubation, cells were mounted in ProLong® Diamond Antifade Mountant with DAPI (Life technologies, USA). Uninfected cells and ZIKV-infected cells stained with mouse isotype IgG2a antibody (Dako, Denmark) were used as negative controls. ZIKV-infected cells were identified by using a Zeiss LSM 700 confocal laser scanning microscope fitted on an Axio observer Z1 inverted microscope (Zeiss, Breda, the Netherlands). All images were processed using Zen 2010 software (Zeiss).