secured along the edges with 4–0 suture. The exposed tissue was superfused with 37uC warmed bicarbonate-buffered saline, pH 7.4 and covered with a coverslip. Intravital visualization of leukocyte biology was performed using an upright microscope. Preparation of the mouse ear for IVM The jugular vein of an anesthetised mouse was cannulated to permit intravenous delivery of antibodies and additional anaes- thetic, if required. The mouse was placed on a heat pad to maintain the body temperature and the dorsal hair from the right ear was gently removed using depilatory cream (Nair; Church & Dwight Co., Inc, Princeton, NJ) without causing any irritation. The ear was carefully flattened out on an elevated preparation board, superfused with 37uC warmed bicarbonate-buffered saline, pH 7.4, and held in place with a coverslip applied on the dorsal side of the ear. Intravital visualization of leukocyte biology was performed using an upright microscope. Image Processing Still images were exported from the Volocity (Improvision) acquisition software as .tif images. Images for platelet aggregate quantification were imported directly into ImageJ. Display items were processed using Photoshop (Adobe, San Jose, CA) to adjust the minimum threshold values for each of the fluorescence channels. The same threshold values were applied to images from all treatment groups within a single experiment. Videos underwent contrast enhancement within the Volocity software package, adjusting the Black Point for each fluorescence channel. Again, the same settings were applied to the videos of all treatment groups within a given experiment. Videos were exported as .avi files and were converted to an appropriate size, resolution, and frame rate using Microsoft Movie Maker (Microsoft Canada, Mississauga, Ontario, Canada) and Prism Video Converter Software (NCH Software Inc., Greenwood Village CO). 3D Model Generation Z Stacks of xy planes (0.5 mm intervals) were imaged using an inverted spinning-disk confocal microscope using either the ASI focus drive (Applied Scientific Instrumentation) or Olympus focus drive (Olympus). 3D isosurface models of platelet aggregates were rendered within the Volocity software package (Improvision) using the 3D-opacity option. The same Black Level settings for each fluorescence channel were applied to all images. For 3D videos, individual frames were rendered using the default photobleaching compensation function within the Volocity software package. Semi-quantitative Analysis of Platelet Aggregates Snapshots were generated from intravital videos and the images corresponding to the red fluorescence channel alone (PE- conjugated anti-CD49b) were exported as .tif documents. For analysis of aggregate size and number, images were opened with the ImageJ software package V1.41 (NIH at http://rsb.info.nih. gov/ij/) and image contrast was set to maximum to sharply define the borders of each platelet aggregate. To account for variability in background fluorescence between experiments and between Ab lot, and to eliminate fluorescence attributed to rapidly circulating platelets, the minimum brightness threshold was adjusted for each image. To determine the threshold value, LPS treated mice were included in all experiments. Threshold values were set to yield an average of 12–14 aggregates $50 mm2 for all fields of view of LPS treated animals. This number was empirically determined through examination of fields of view from LPS treated animals and enumeration of platelet aggregates visualized to be the size of, or Intravital Visualization of Platelets larger than neutrophils. Once the threshold value was determined for each experiment, this value was then applied to images from all treatment groups within the experiment, thereby allowing for direct comparison of aggregate sizes and number between treatment groups. Once the minimum brightness threshold was set for each image, the number of platelet aggregates $10, 25, 50, or 100 mm2 were counted using the Analyze Particles function within ImageJ. Statistical Analysis The number of platelet aggregates within each size category (Fig. 2D, S5) was compared between treatment groups using a one-way analysis of variance (ANOVA) with a Bonferroni post-test for multi-group comparison. Untreated (n = 3), LPS treated (n = 5), anti-CD18 + LPS treated (n = 4), neutrophil depleted + LPS (n = 5), Kupffer cell depleted + LPS (n = 4) neutrophil and Kupffer cell depleted + LPS (n = 3); a minimum of 5 fields of view were analyzed for each animal. Platelet counts (Fig. S2) between untreated and LPS treated mice were compared using an unpaired Student’s t-test. Untreated (n = 5), LPS treated (n = 4). Comparisons of the number of adherent platelets, interacting platelets (Fig. 3C), and platelets bound per neutrophil (Fig. 3D) between untreated and LPS-treated mice were made using unpaired Student’s t-tests. Comparison of the percentage of neutrophils with either 0 or 3 platelets bound (Fig. 3E) in the untreated and LPS treated groups was made using a two-way ANOVA with a Bonferroni post-test for multi-group comparison. Untreated (n = 4), LPS treated (n = 5); a minimum of 4 fields of view were analyzed for each animal. Comparison of the number of platelets bound to neutrophils, KC, and sinusoids (Fig. 4C) between untreated and LPS treated mice was made using a two-way ANOVA with a Bonferroni post- test for multi-group comparison. Untreated (n = 4), LPS treated (n = 5); a minimum of 4 fields of view were analyzed for each animal. Comparisons in the total area of neutrophil and Kupffer cell staining (Fig. S7) between untreated and LPS treated mice were made using a two-way ANOVA with a Bonferroni post-test for multi-group comparison. Area of staining was measured in 4 mice in each treatment group (2–4 FOV/mouse). Supporting Information Figure S1 Intravital visualization of platelets labelled with PE-conjugated anti-CD49b within the mouse ear. Spinning-disk confocal-microscopic imaging of mouse ear micro- vasculature in untreated mice. Visualization of endothelium with Alexa Fluor 488-conjugated anti-CD31 (i), neutrophils labelled with Alexa Fluor 647-conjugated anti-Gr-1 (ii), platelets stained with PE-conjugated anti-CD49b (iii), and multi-channel overlay (iv). No adherent and few endothelial-interacting platelets were seen in the mouse ear, a tissue visualized without surgical intervention, under control conditions. All scale bars, 20 mm. (TIF) Figure S2 In vivo labelling of mouse NK cells by intravenous injection of the anti-CD49b antibody HMa2. Intravenous administered PE-conjugated anti-CD49b (red) also labels NK cells in vivo (white arrow). Their larger size and less intense staining makes labelled NK cells easily discernable from nearby platelets (small yellow arrows). Neutrophils are labelled with Alexa Fluor 647-conjugated anti-Gr-1 (blue); green is liver autofluorescence to illustrate vessels. Scale bar, 20 mm. (TIF) PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e25109