saline injected intrascrotally 4 h prior to visualization. Neutrophil depletion was performed by i.p. injection of 200 mg of unconju- gated GR-1 24 h prior to LPS treatment. Kupffer cell depletion was achieved by i.v. injection of 200 ml of clodronate liposomes 30 h prior to LPS treatment as previously described [49,50]. Flow Cytometry Approximately 900 ml of blood was collected from anesthetised mice via cardiac puncture into a syringe containing 100 ml of acid- citrate dextrose. For platelets, blood was centrifuged for 10 min at 1006g at room temperature in a desktop microfuge and the platelet rich plasma was collected. Collected plasma was centrifuged a second time at 1006g 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 ml of cold FACS wash buffer. For leukocytes, whole blood was lysed in a 36 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). Platelet Counts Approximately 450 ml of blood was collected from anesthetised animals via cardiac puncture into a syringe containing 50 ml 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 106/ 0.40 and UPLANSAPO 206/0.70 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 UP- LANFL N 106/0.30W and XLUMPPlanFI 206/0.95W objec- tive 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 Intravital Visualization of Platelets filters (Semrock, Rochester, NY) driven by a MAC 6000 Modular Automation Controller (Ludi Electronic Products, Ltd., Hawthorne, NY) and detected with a 5126512 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 [51]. 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 [52]. 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 PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e25109