Histones uncovered during the breakdown of the first wave of NETs contribute to the second wave of the traps. To estimate the total volume of NET proteins remaining on the endothelium and outside phagocytes, their 3D models rendered from z-stacks were obtained and quantified with IVM. The changes were followed during days 13 and 14 after induction of endotoxemia (LPS IP; 1 mg/kg b.w.), and they were compared with the first 6 hours (high NET levels) and day 10 (low NET levels). The standard imaging time was 10 AM, but on day 13, data were also collected 9 and 17 hours later (day 13 + 9 hours and day 13 + 17 hours, respectively). (A-B) Total volume of H2A.X histones (A) and NE (B) was thus estimated, independently of their location against each other (outside or hidden). Then using a designated mask, only H2A.X histones exposed over NE were measured (C) and vice versa for NE; that is, only NE exposed over histones was quantified (D). Concomitantly, soluble H2A.X (E) and sNE (F) were estimated in blood. (G-H) Representative images of 3D models of histones (red) and NE (violet) within the NET structure attached to the liver endothelium. (G) The presence and position of histones and NE against each other (upper), and the amount of histones exposed to the lumen (lower). (H) The inserts from panel G are enlarged to indicate the difference between the amount of exposed H2A.X histones over NE between different time points during day 13 after endotoxemia. The scale bar indicates 20 μm. The data in the graphs are expressed as the mean ± SD of at least 3 fields of view. Statistically significant differences according to the 1-way ANOVA are designated by letters, in which the same letter indicates no differences between groups, and different letters indicate statistical differences (Bonferroni post hoc; n = 3-5). sNE, soluble NE.