Immunofluorescent Staining of ZIKV Envelope Protein

To monitor ZIKV infection in cells, supernatant was removed at the indicated times and cells were rinsed in PBS. Cells were fixed in PBS–4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) for 15 min at room temperature (RT), and permeabilized with PBS–0.1% Triton (Sigma) for 3 min at RT. Non-specific sites were blocked with PBS–1% bovine serum albumin (BSA, Sigma)–0.1% Tween 20 (Sigma) for 30 min at RT. ZIKV envelope protein (E) staining was performed using a primary mouse anti-flaviviral E antibody (4G2; home-purified from the ATCC hybridoma [30 (link)]) diluted in PBS–0.2% BSA–0.2% Tween 20 for 1 h at RT, and a secondary Alexa Fluor 488-coupled goat anti-mouse antibody (Life technologies) diluted in PBS–0.2% BSA for 30 min at RT. Finally, cells were mounted in Fluoromount G–DAPI (SouthernBiotech, Birmingham, AL, USA) and imaged on a fluorescence microscope (EVOS FL, Life Technologies). Cells were washed twice with PBS between each step.