Generation and analysis of an acute platelet-depletion model induced by single high-dose injection of an anti-GPIbα antibody. (A) An anti-GPIbα antibody (5A7 or R300, 2 mg/kg) was administered to C57BL/6J wild-type (WT) mice by retro-orbital injection, and platelet counts were monitored. (B-C) Platelet distribution was visualized by wide-field immunofluorescence analysis of cryosections prepared from spleen (B) and liver (C) harvested from untreated mice or from treated mice 30 minutes or 24 hours after antibody administration (BZ-X700 microscope). Bars represent 100 µm. Tissues were stained with DAPI (blue), anti-rat IgG (green; detecting administered 5A7 antibody), and anti-αIIb antibody (purple). (D) Immunofluorescence staining of cryosectioned liver. Opsonized platelets were stained with anti-rat IgG (green) and macrophages were stained with anti-F4/80 antibody (red). Hepatocytes were stained with anti-ASGPR1 antibody (red; bottom). Images were obtained by confocal microscopy (LSM 880 microscope). Bars represent 20 µm. (E) IVIG was administered via intraperitoneal injection (2 g/kg) 24 hours before 5A7 IV injection (0.6 mg/kg). Spleens harvested 20 minutes after 5A7 administration were cryosectioned and immunostained with anti-rat IgG (green; detecting administered 5A7 antibody) and anti-F4/80 antibody (red), along with costaining with DAPI (blue). (F) Images were binarized, and colocalization of opsonized platelets (stained by anti-rat IgG) with F4/80 was calculated as a ratio by quantifying anti-rat IgG and F4/80 costained area divided by total of anti-rat IgG–stained area, by using Fiji image-analysis software. ***P < .001 by unpaired Student t test.