All experiments involving animals were approved by the University of Calgary Animal Care Committee (Protocol # MO8131) and conform to the guidelines established by the Canadian Council for Animal Care. For all experiments, mice were anesthetised by intra-peritoneal (i.p.) injection of 200 mg/kg ketamine (Bayer Inc Animal Health, Toronto, Ontario, Canada) and 10 mg/kg xylazine (Bimeda-MTC, Cambridge, Ontario, Canada).
Wildtype C57BL/6 (The Jackson Laboratory, Bar Harbor, ME) and CD41-YFPki/+ (a generous gift from Dr. Kelly McNagny, University of British Columbia, Vancouver, British Columbia, Canada) colonies were maintained in specific-pathogen free facilities at the University of Calgary. At the time of use, animals were between 7 and 10 weeks of age and weighed 20–30 g.
Antibodies and Treatments
Phycoerythrin (PE)-conjugated and allophycocyanin (APC)-conjugated Armenian hamster anti-mouse CD49b (clone HMa2), PE-conjugated rat anti-mouse CD41 (clone MWReg30), and FITC-conjugated goat-anti-rat IgG were purchased from BD Biosciences Pharmingen (San Diego, CA). PE-conjugated rat anti-mouse F4/80 (clone BM8), unconjugated and Alexa Fluor 647-conjugated rat anti-mouse Ly-6G (GR1) (clone RB6-8C5), unconjugated rat anti-mouse CD18 (clone GAME-46), unconjugated rat anti-mouse PECAM-1 (clone 390), and unconjugated rat IgG1 isotype control antibodies were purchased from eBioscience (San Diego, CA). For intravital microscopy, rat anti-mouse PECAM-1 mAb was conjugated to Alexa Fluor 488 using a protein labelling kit as per the manufacturer's instructions (Invitrogen, Eugene, OR). In vivo
blockade of CD18 was achieved by i.v. administration of 100 µg of anti-CD18 mAb 20 min prior to LPS treatment. Platelet depletion was performed by i.p. injection of 100 µl of anti-thrombocyte serum (Cedarlane, Burlington, Ontario, Canada). Highly purified lipopolysaccharide (LPS) (Escherichia coli O111
B4) was purchased from Calbiochem (EMD Sciences, San Diego, CA). For liver and brain studies, mice were treated with 1 mg/kg LPS i.v. 4 h prior to intravital analysis; in cremaster studies, mice received 0.5 µg/kg LPS in 200 µl of saline injected intrascrotally 4 h prior to visualization. Neutrophil depletion was performed by i.p. injection of 200 µg of unconjugated GR-1 24 h prior to LPS treatment. Kupffer cell depletion was achieved by i.v. injection of 200 µl of clodronate liposomes 30 h prior to LPS treatment as previously described 
Approximately 900 µl of blood was collected from anesthetised mice via cardiac puncture into a syringe containing 100 µl of acid-citrate dextrose. For platelets, blood was centrifuged for 10 min at 100×g at room temperature in a desktop microfuge and the platelet rich plasma was collected. Collected plasma was centrifuged a second time at 100×g for 3 min to remove any contaminating red blood cells. Platelet rich plasma was collected into a new microfuge tube and stained with mAb diluted in FACS wash buffer (PBS containing 1 mM EDTA, 2% FBS) for 30 min on ice. Platelets were washed with cold FACS wash buffer, pelleted by centrifugation, resuspended in 100 µl of cold FACS wash buffer. For leukocytes, whole blood was lysed in a 3× volume of ACK Lysing Buffer (Lonza, Walkersville, MD), pelleted by centrifugation, washed with cold FACS wash buffer. Cells were stained with mAb diluted in FACS wash buffer for 30 min on ice, washed and resuspended in cold FACS wash buffer. Analysis was performed on either a FACScan (Beckton Dickinson, Mississauga, Ontario, Canada) or Attune Acoustic Focusing Cytometer (Life Technologies, Carlsbad, California).
Approximately 450 µl of blood was collected from anesthetised animals via cardiac puncture into a syringe containing 50 µl of 0.5 M EDTA. Blood was transferred to EDTA coated microtainer tubes (Beckton Dickinson). Samples were transported to Calgary Lab Services for haematological analysis.
Spinning-disk Confocal Intravital Microscopy (IVM)
Two different spinning-disk confocal microscopes were used in these studies.
Liver microscopy was performed using an Olympus IX81 inverted microscope (Olympus, Center Valley, PA), equipped with an Olympus focus drive and a motorized stage (Applied Scientific Instrumentation, Eugene, OR). This microscope is fitted with a motorized objective turret equipped with UPLANSAPO 10×/0.40 and UPLANSAPO 20×/0.70 objective lenses and is mounted to an optical table (Newport, Irvine, CA) to minimize vibration when imaging. This microscope is coupled to a confocal light path (WaveFx; Quorum Technologies, Guelph, Ontario, Canada) based on a modified Yokogawa CSU-10 head (Yokogawa Electric Corporation, Tokyo, Japan).
Brain, cremaster, and ear microscopy was performed using an Olympus BX51WI upright microscope (Olympus), equipped with a Ludl focus drive and a motorized stage (Applied Scientific Instrumentation). This microscope is mounted to a optical breadboard (Newport) to minimize vibration, fitted with UPLANFL N 10×/0.30W and XLUMPPlanFI 20×/0.95W objective lenses, and is coupled to a confocal light path (WaveFx; Quorum Technologies) based on a modified Yokogawa CSU-10 head (Yokogawa Electric Corporation).
For both microscopes, each of 491-, 561-, and 642-nm excitation laser wavelengths (Cobolt, Stockholm, Sweden) were sequentially controlled and merged into a single optic cable using an LMM5 laser merge module (Spectral Applied Research, Richmond Hill, Ontario, Canada). Fluorescence was visualized through one of ET 525/50M (green channel), FF 593/40 (red channel), or ET 700/75M (far red channel) band pass emission filters (Semrock, Rochester, NY) driven by a MAC 6000 Modular Automation Controller (Ludi Electronic Products, Ltd., Hawthorne, NY) and detected with a 512×512 pixels back-thinned EMCCD camera (C9100-13, Hamamatsu, Bridgewater, NJ). Volocity Acquisition software (V5.2.1 – Inverted; V4.3.2 – Upright) (Improvision Inc., Lexington, MA) was used to drive the confocal microscope. Image acquisition settings varied according to the microscope used and the tissue visualized. Typical laser power, exposure time and sensitivity settings are as follows, Liver (inverted); green channel (autofluorescence –80%, 415 ms, 215; CD41-YFP –75%, 425 ms, 250), red channel (CD49b –90%, 150 ms, 210; F4/80–90%, 100 ms, 210), far red channel (Ly-6G –80%, 300 ms, 200), Brain (upright); green channel (PECAM-1–95%, 300 ms, 240), red channel (CD49b –100%, 70 ms, 240), far red channel (Ly-6C –100%, 300 ms, 250), Cremaster and Ear (upright); green channel (PECAM-1–80%, 500 ms, 115), red channel (CD49b –95%, 500 ms, 120), far red channel (Ly-6G –80%, 500 ms, 120). Green, red, and far red channels were overlaid using brightest point settings before exporting in .tiff or .avi format.
Preparation of the Mouse Liver for Intravital Microscopy
Intravital microscopy of the mouse liver was performed as previously described 
. Briefly, the tail vein of anesthetised mice was cannulated to permit the delivery of fluorescently labelled Ab and for maintenance of anesthetic. Mouse body temperature was maintained using an infrared heat lamp. A midline incision followed by a lateral incision along the costal margin to the midaxillary line was performed to expose the liver. The mouse was place in a right lateral position and the ligaments attaching the liver to the diaphragm and the stomach were cut allowing the liver to be externalized onto a glass coverslip on the inverted microscope stage. Exposed abdominal tissues were covered with saline-soaked gauze to prevent dehydration. The liver was draped with a saline soaked KimWipe to avoid tissue dehydration and to help restrict movement of the tissue on the slide.
Preparation of the mouse brain for IVM
The tail vein of an anesthetised mouse was cannulated to permit intravenous delivery of antibodies and additional anesthetic, if required. To isolate movement, the animal's head was held in a stereotaxic board. Skin covering the parietal bone of the mouse skull was reflected and the left parietal bone was carefully thinned using a high-speed drill (Fine Science Tools, North Vancouver, British Columbia, Canada) to allow for transillumination. Care was taken as to not open the cranial vault to ensure physiological pressures and blood flow were maintained. Intravital visualization of leukocyte biology within the pial microvasculature was performed through this thinned skull tissue using an upright microscope.
Preparation of the mouse cremaster for IVM
The mouse cremaster muscle was used to study neutrophil recruitment as previously described 
. In brief, the jugular vein of an anesthetised mouse was cannulated to permit intravenous delivery of antibodies and additional anaesthetic, if required. Mice were placed on a special cremaster preparation board and body temperature was maintained through a heat pad. An incision was made in the scrotal skin to expose the left cremaster muscle, which was then carefully dissected free of the associated fascia. The cremaster muscle was cut longitudinally with a cautery. The testicle and the epididymis were separated from the underlying muscle and were moved into the abdominal cavity. The muscle was held flat on an optically clear viewing pedestal and was secured along the edges with 4–0 suture. The exposed tissue was superfused with 37°C 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 anaesthetic, 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 37°C 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.
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 µm 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 µm2
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 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 µm2
were counted using the Analyze Particles function within ImageJ.
The number of platelet aggregates within each size category (, 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
Comparisons of the number of adherent platelets, interacting platelets (), and platelets bound per neutrophil () 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 () 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 () 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).