Spatial relationship between thrombin activity and NETs in the microvasculature. (A) Representative SD-IVM images of the liver microcirculation of untreated and LPS-treated mice. Thrombin probe fluorescence is in green, NETs are shown in red (AF555 anti-H2Ax), and neutrophils are shown in blue (AF647 anti-Gr1). Bars represent 50 μm. (B) Magnified inset showing a liver sinusoid containing an adherent neutrophil (blue), surrounding NETs (red), and the associated thrombin activity (green). (C) The regions of the highest thrombin probe signal within each liver sinusoid (per field of view) were identified in LPS-treated mice and categorized according to the spatial relationship with the nearest NET relative to the direction of blood flow: downstream, within NET, upstream, or not associated (>100 μm from the nearest NET). Data are represented as mean ± SEM. *P < .05; N = 4 mice per group.