Dapi nucleic acid stain

For visualization of EGFP-tagged constructs, cells grown on glass coverslips were fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS) at 4°C for 10 min, then washed with PBS and counterstained with DAPI Nucleic Acid Stain (Invitrogen). For ubiquitin labeling, N2a cells were fixed in methanol for 10 min at −20°C, re-hydrated with PBS for 10 min and blocked for 30 min in 5% (w/v) bovine serum albumin with 0.3% (w/v) Triton X-100 in PBS at room temperature. Immunocytochemistry was performed using monoclonal antibody recognizing ubiquitin (mAb1510; Chemicon) at 1:1000 diluted in PBS. Antibody distribution was visualized by epifluorescence microscopy after incubation with secondary antibody conjugated to Alexa 594 diluted at 1:350 in PBS (Molecular Probes), and finally counterstained with DAPI Nucleic Acid Stain. Labeled cells were visualized using a Leica DM6000 microscope and digital images captured using Volocity imaging software (PerkinElmer).

Immunostaining and confocal microscopy were performed as we previously described [32 (link),33 (link)]. Briefly, MDMs and moDCs cultured in 24-well plate were digested by trypsin and re-seeded into chamber slide. After one day of culture in the RPMI-1640 with growth factors, the cells were uninfected or infected with ZIKV^PR or ZIKV^U (MOI=1.0) for 2 h at 37°C. At 48hpi, the infected MDMs and moDCs were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde. The cells were permeabilized for 10 min with 0.2% Triton X-100 and blocked with Dako blocking buffer for 30 min at room temperature. The cells were subsequently incubated with mouse 4G2 monoclonal antibody (MERCK) against envelope (E) protein of pan-flavivirus or mouse J2 monoclonal antibody (SCICONS) against double-stranded RNA (dsRNA) diluted with Dako antibody diluent overnight at 4°C, followed by incubation with goat anti-mouse Fluor 488 immunoglobulin G (IgG) (Abcam) secondary antibody diluted with Dako antibody diluent for 1 h. The nuclei of the cell were stained by DAPI nucleic acid stain (Thermo Fisher Scientific) for 10 min. The slides were imaged with confocal microscopy using a Carl Zeiss LSM 880 system (Zeiss, Oberkochen, Germany).