Dapi fluoromount g

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.

To assess ZIKV infection in cells, supernatant was removed at the indicated times and cells were rinsed in PBS. Then, cells were fixated in a 4% paraformaldehyde (PFA) solution for 15 min at RT and permeabilized with PBS – 0.2% Triton for 5 min at RT. Then, ZIKV envelope protein (E) staining was performed overnight at 4°C using a primary mouse anti-E antibody (4G2), and for 1 h at RT with a secondary Alexa Fluor 488-coupled goat anti-mouse antibody (Life technologies). Finally, cells were mounted in Fluoromount G – DAPI (SouthernBiotech) and imaged on a fluorescence microscope (EVOS FL, Life Technologies).
Presence of tight junctions in Caco-2 monolayers was assessed by zonula occludens-1 (ZO-1) immunostaining. Cells were fixed in PBS – 80% methanol (Sigma) for 15 min at RT and permeabilized with PBS – 0.2% Triton for 5 min at RT. Non-specific sites were blocked with PBS – 5% BSA for 30 min at RT. ZO-1 was stained by incubating cells with a polyclonal rabbit anti-ZO-1 (Invitrogen) overnight at 4°C as primary antibody, and an Alexa Fluor 546-coupled donkey anti-rabbit (Invitrogen) for 1 h at RT as secondary antibody, using the same buffers as previously (Hubert et al., 2019 (link)). Finally, cells were mounted in Fluoromount G – DAPI and imaged on the same fluorescence microscope as above. Cells were washed twice with PBS between each step.

As previously described, a cell migration assay on cardiomyoblast cell line (H9C2) was performed [3 (link)]. Briefly, a confluent monolayer of H9C2 cells (CRL-1446, ATCC, Manassas, VA, USA) was scratched with a 200 µL pipette tip, then the cells were washed in PBS and incubated with 5 × 10⁸ EVs/mL in 1% EV-depleted (fetal bovine serum) FBS medium for 24 h, and PBS was used as negative control. The cell cultures were fixed in 4% formaldehyde and stained with Fluoromount g (DAPI, SouthernBiotech Birmingham, Al, USA). An EVOS cell imaging system (Thermo Fisher Scientific, Waltham, MA, USA) was used to take digital images of the stained cell cultures. ImageJ Freeware (imagej.nih.gov/version 1.8.0, National Institutes of Health, Bethesda, MD, USA) was used to quantify differences in the scratch areas between the experimental groups.

Mice were perfused and brains were cryopreserved and 30–40 μm thick slices were prepared and stained as described previously [58 (link)]. Slices were mounted using Fluoromount G DAPI (Southern biotech).