Mice were anesthetized with a mixture of ketamine/xylazine as described above, and then cannulation of the right jugular vein was performed. Preparation of the liver for intravital imaging was performed as previously described by Kolaczkowska et al. [51 (link)]. Spinning-disk confocal intravital microscopy was performed using ZEISS Axio Examiner.Z1 upright microscope equipped with confocal spinning disk device DSD2 (described above). The following filters were used: four excitation filters (DAPI: 390/40 nm; GFP: 482/18 nm; RFP: 561/14 nm; Cy5: 640/14 nm) and appropriate emission filters (DAPI: 452/45 nm, exposure time 600 ms; GFP: 525/45 nm, exposure time 700 ms; RFP: 609/54 nm, exposure time 500 ms; Cy5: 676/29 nm, exposure time 900 ms). Following antibodies were used to detect presence of NETs and neutrophils in the liver vasculature: Alexa Fluor 647 antineutrophil elastase (1.6μg/mouse, clone G-2; Santa Cruz Biotechnology, Dallas, TX, USA), histone H2A.X (0.5µg/mouse, clone 938CT5.1.1; Santa Cruz Biotechnology, Dallas, TX, USA), Brilliant Violet 421 anti-Ly6G (1.6 μg/mouse, clone 1A8; BioLegend, San Diego, CA, USA). All antibodies were injected intravenously (i.v.) via the jugular vein ~20 min prior to intravital imaging as previously published methodology [14 (link)].
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