PMN traversal across TNFα-stimulated PCPECs. PCPEC monolayers were stained for ZO-1 (red), actin (phalloidin, green), nuclei (DAPI, blue) and PMNs (SWC3a-FITC, yellow). Three-dimensional immunofluorescence images of TNFα-stimulated 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) PMN located below a multicellular corner with a gap in the ZO-1 structure (white arrow). (F-J) Transcellular (central PMN) PMN and migration close to TJ (left PMN). The right PMN is located directly beyond an intact TJ strand, the left one is close to disrupted ZO-1 (white arrow). (K-O) PMN migration close to an interrupted TJ strand (white arrow). This figure is a representative example of three independent experiments that all gave similar results. Scale bar, as indicated.