Polyclonal rabbit anti zo 1

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.

All primary and secondary antibodies were dissolved in PBS (1:200) prior to labeling. The PBECs, astrocytes and pericytes were fixed in 4% paraformaldehyde and blocked in PBS supplemented with 0.2% Triton-X-100 and 3% bovine serum albumin for 1 hour. The PBECs were stained with polyclonal rabbit anti-claudin-5 (Sigma-Aldrich, cat. no. SAB4502981, lot 310145) and polyclonal rabbit anti-ZO-1 (Invitrogen, cat. no. 617300, lot 1087989A). Mixed glial cells were stained with rabbit anti-glial fibrillary acidic protein (GFAP)(DAKO, DK, cat. no. Z0334, lot 20003791) and Texas Red labelled Lycopersicon Esculentum (Tomato) Lectin (Vector Labs, Peterborough, United Kingdom, cat. no. TL1176, lot W0812). Pericytes were stained with monoclonal mouse anti-α-smooth muscle actin (α-SMA) (Sigma-Aldrich, cat. no. A5228, lot 091M4832), polyclonal rabbit anti-ZO-1 and rabbit anti-platelet-derived growth factor receptor-beta (PDGFR-β) (Santa Cruz, cat.no.Sc-432, lot K1113). For detection, the cells were subsequently stained with goat anti-rabbit Alexa 488 or goat anti-mouse Alexa 585 (Invitrogen) as the secondary antibodies. All cells were counterstained with DAPI. The Millicell membranes were cut out of the inserts and mounted on glass slides in fluorescent mounting media (Dako, Denmark) and cover slips were placed upon the membranes.

IEC-6 cells in chamber slides were fixed with 1% paraformaldehyde in PBS for 10 min at room temperature. After washing in PBS and blocking nonspecific binding sites with 5% bovine serum albumin (BSA), the slides were incubated with polyclonal rabbit anti-ZO-1 (5 mg/ml, Invitrogen) in PBS with 5% bovine serum albumin (BSA) for 120 min at room temperature. After washing, the slides were incubated with rhodamine phalloidin (Invitrogen) and DyLightTM 488-conjugated AffiniPure Donkey anti-rabbit IgG (0.075 mg/ml, Jackson Lab, WestGrove, PA) for 60 min. The slides were then washed and mounted with coverslips using ProLong Gold anti-fade reagent with DAPI (Invitrogen). The slides were imaged with a confocal microscope (Nikon).