The location of PMN adhesion and subsequent transmigration on ICAM-1GFP iHUVEC monolayers. (A) The initial adhesion sites of PMNs and the sites of TEM were determined from review of individual images using ImageJ, and these positions were overlaid onto the VE-cadherin staining pattern graph obtained prior to introduction of PMNs into the chamber. Bar, 20 μm. (B) The transmigrated PMNs were grouped according to their sites of initial arrest and TEM at junction or nonjunction (> 5 μm from any junction). The fraction of PMNs in each group was calculated. ^*P < .05. The black and gray error bars correspond to paracellular and transcellular routes, respectively. (C) Increasing numbers of PMNs (as indicated) were drawn across 4-hour TNF-α iHUVEC monolayers at 0.5 dyne/cm². Initial location of arrested PMNs (left) and the route of TEM (right) were determined as detailed in “Materials and methods.”