Distribution of the NiV glycoproteins and the NiV receptor EB2 on the surface of polarized endothelial cells. PBMEC (A) and PAEC-EB2 (B and C) were cultured on filter supports for 6 or 5 days, respectively. (A, B) Polarized cell cultures were infected with NiV at a m.o.i. of 0.5. At 24 h p.i., cells were inactivated and fixed with 4% PFA and then incubated from both sides with monoclonal antibodies directed either against the F or the G protein, followed by incubation with AlexaFluor 568-conjugated secondary antibodies. Confocal horizontal (xy) sections through the apical part of the cell monolayer are shown in the left panel. White lines indicate the area along which vertical sections were recorded. Vertical (xz) sections through the foci are shown on the left panel. (C) Cells were fixed and surface-stained from both sides with a EB2-specific ligand (EphB4/Fc) and a AlexaFluor 568-labelled secondary antibody. Then cells were permeabilized and incubated with a VE-cadherin specific antibody and a AlexaFluor 488-conjugated secondary antibody. Confocal horizontal (xy) and vertical (xz) sections are shown.