JAQ1-opsonized platelets are “trapped” primarily in the liver of WT, but not FcγRIIB-deficient mice. (A) Sequestration of fluorescently labeled platelets was monitored in anesthetized mice upon injection of JAQ1 or vehicle using an in vivo imaging system. (B) Sections of snap-frozen liver samples of WT mice and mice lacking FcγRIIB (Fcgr2b^−/−) were probed with horseradish peroxidase–conjugated anti-GPIb antibodies. Detection was performed using 3-Amino-9-ethylcarbazole. (C-H) For confocal microscopy, liver sections of WT mice treated with DyLight488-conjugated JAQ1 (green) 30 min before organ extraction were stained for platelets (anti-GPIX, light blue), Kupffer cells (F4/80, blue), and endothelium (anti-CD105, red), and counterstained by 4′,6-diamidino-2-phenylindole (gray). (C) Platelets per visual field (388 × 388 µm) were quantified in liver sections of WT and FcgR2b^−/− mice (4-5 sections per animal, n ≥ 3; n.d., not detectable). (D) Platelets (light blue) can be detected in close proximity to the endothelium (white arrows) or Kupffer cells (green arrows). Scale bar, 20 µm. (E) Platelet counts of mice treated with clodronate liposomes (clodronate) or PBS liposomes (vehicle) 48 h before the experiment were monitored upon injection of the anti-GPVI antibody JAQ1^biotin by flow cytometry. (F) Platelets per visual field were quantified in liver sections of clodronate and vehicle liposome-treated mice (4-5 sections per animal, n = 3). (G) JAQ1^Dy488-clusters per visual field (388 × 388 µm) were quantified in liver sections of WT and FcgR2b^−/− mice (4-5 sections per animal, n ≥ 3; n.d., not detectable). (H) JAQ1^Dy488 (green, left panel = isolated channel, right panel = merge) can be detected in WT liver sections attached to some platelets (light blue arrow), engulfed by Kupffer cells (green arrows) or endothelial cells (white arrows). Scale bar, 10 µm.