Intravital confocal microscopy reveals reduced LPS-induced neutrophil sequestration in anti-GPVI–treated mice. (A) Experimental scheme of the model of LPS-induced ALI in C57BL/6J WT mice treated with either 4 mg/kg bw anti-GPVI antibody JAQ1, or nonimmune IgG (control) IP, 5 days before intravital confocal microscopy. LPS was administered at a dose of 5 mg/kg bw IN. Imaging was performed with a TCS-SP8 confocal laser scanning microscope (Leica Microsystems) with a 25× objective (HC FLUOTAR L 25×/0.95 WATER, Leica Microsystems) after 1 hour, and mice received fluorescently labeled antibody derivates to label neutrophils (Ly6G⁺, yellow), platelets (GPIX⁺, cyan), and vessels (CD31⁺, magenta). After 5 minutes, the FOV was changed. Total duration of imaging was 60 minutes. The time intervals from 0 to 5 minutes, 25 to 30 minutes, and 55 to 60 minutes were used for quantification. (B) Representative images derived from videos of the time interval from 25 to 30 minutes, illustrating the difference in neutrophil and platelet abundance in the 2 groups. Scale bar, 30 μm. (C) Quantification of the area (μm²) covered by the neutrophil (top graph) and platelet (bottom graph) fluorescence signal analyzed with Imaris Bitplane software at the indicated time intervals. (D) Automated tracking of neutrophils with Imaris Bitplane software. Representative images derived from videos of the time interval from 25 to 30 minutes showing probability masks of neutrophils superimposed with neutrophil tracks (left image) or the neutrophil tracks only (right image) of the indicated group. Blue tracks, adhesive neutrophils; red tracks, tethering neutrophils; and white tracks, crawling neutrophils. Scale bar, 30 μm. (E-G) Fraction of total neutrophils that were adhesive (E), tethering (F), and crawling (G). Each data point represents 1 mouse at the respective time interval. Bars represent mean time interval ± standard deviation. The two-way ANOVA with a Tukey multiple comparisons test. ∗P ≤ .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.