PMN traversal across inverted control PCPEC cultures. PCPEC monolayers were stained for ZO-1 (red), actin (phalloidin, green), nuclei (DAPI, blue) and PMNs (SWC3a-FITC, yellow). Three-dimensional immunofluorescence images of control PCPECs co-cultured with PMNs were reconstructed from 0.3 μm Apotome® optical sections, using Zeiss software Inside 4D. Views from above (on the apical cell side) show different phases of PMN migration. The lower row represents side views (D, E, I, J, N, O). A-E. Transcellular PMN migration in distance to ZO-1. F-J. PMN below tight junctions and actin ring. K-O. PMN migration stops below altered tight junctions with altered ZO-1 strands (white arrows). This figure is a representative example of three independent experiments that all gave similar results. Scale bar, as indicated.