Analysis of BM MKs in the acute platelet-depletion model. (A-C) BM sections harvested at the indicated time points after IV 5A7 injection were stained with DAPI (blue), anti-rat IgG (green; detecting administered 5A7 antibody), and anti-αIIb antibody (purple) and visualized by BZ-X700 microscope (bars represent 100 µm) (A) or confocal microscopy (LSM 880 microscope; bars represent 10 µm) (B). (C) Images were binarized, and colocalization of 5A7 with integrin αIIb was calculated as a percentage by quantifying the anti-rat IgG–stained area and dividing by the anti-integrin αIIb antibody–stained area, by using Fiji image-analysis software. ***P < .001 by one-way analysis of variance with Dunn’s multiple-comparison test. (D) BM cells were harvested 24 hours after IV 5A7 injection and analyzed by FACS for GPIbα expression by staining with monoclonal anti-GPIbα antibody (Xia.G5) which does not compete with 5A7 for binding to GPIbα. *P < .05, ***P < .001 by 2-way analysis of variance with Sidak’s multiple-comparison test.