Hb 112

Inserts were washed with PBS and incubated in 4% paraformaldehyde (PFA) for 15 min. In a second step, the inserts were incubated in confocal buffer (50 nM ammonium chloride, 0.1% saponin in PBS) for 30 min. Cells were incubated with anti-Zonula occludens 1 (ZO-1, clone 1A12, Thermofisher) and anti-E protein Flavivirus group antibody 4G2 (ATCC, HB-112™) for 2 h followed by the Alexa 633 coupled goat anti-rabbit IgG (Thermofisher), Alexa 488 coupled anti-IgG2a (molecular probes), and anti-β Tubulin directly coupled with Cy3 (clone TUB 2.1, Abcam) in the dark. DAPI (Sigma) was applied to the insert for 5 min and washed with PBS. Each step was performed at room temperature. The membranes were mounted on glass slides in MOWIOL® 4-88 Reagent (Sigma-Aldrich). For confocal microscopy analysis, a Nikon confocal microscope A1 (Nikon) combined with an ECLIPSE Ti inverted microscope (Nikon) and a digital imaging Nikon software (NIS-Elements AR 3.30.02) was used. All images were acquired with the 40X objective and sequential channel acquisition was performed. The images were analyzed with Imaris 8.0.2 software (Bitplane AG, Zurich, Switzerland). To avoid false-positive emissions, different settings were applied including background subtraction, threshold applications, gamma correction, and maxima.