Fig. 3

Immunocytochemical analysis of tight junction and adherens junction proteins 4 days after subculture. a–h Fluorescence images of iBMECs cultured in Thincert inserts. a, b Low magnification images of claudin-5 staining on CN IV-FN (a) and LN 511-E8 (b). Scale bars indicate 200 μm. In (a), examples of regions with internalized claudin-5 are outlined in white, while regions with low junctional expression of claudin-5 are outlined in yellow. c, d Higher magnification images of claudin-5 staining on CN IV-FN (c) and LN 511-E8 (d). Scale bars indicate 25 μm. The images in (c) and (d) are from regions with low claudin-5 expression. e, f Low magnification images of ZO-1 staining on CN IV-FN (e) and LN 511-E8 (f). Scale bars indicate 200 μm. g, h Higher magnification images of ZO-1 staining on CN IV-FN (g) and LN 511-E8 (h). Scale bars indicate 25 μm. Note that for (a–h) the samples are double-stained, so claudin-5 and ZO-1 images are shown for the same cells in each group. i Immunostaining of junctional proteins for iBMECs cultured on ibidi μ-slides coated with CN IV-FN (top row) or LN 511-E8 (bottom row). Claudin-5 staining on CN IV-FN shows a region with significantly internalized claudin-5. White arrows on occludin and ZO-1 images indicate examples of frayed or jagged junctions. Scale bars indicate 50 μm. All images in (a–i) are maximum intensity projections of confocal z-stacks