such as CD41-YFPki/+ mice need to be bred onto other genetic backgrounds. Finally, HMa2 does not interfere with the ability of platelets to bind fibrinogen [44], an important property if one is to examine the role of platelets in inflammation. Using this technique, we have been able to study in vivo, as one example, the formation of platelet aggregates in response to LPS exposure. Furthermore, as all platelets are labelled using this procedure, it is possible to measure the size of individual platelet aggregates and to visualize platelet-platelet interactions. These observations are not possible if only a fraction of platelets are labelled, as is the case in experiments using adoptive transfer of labelled platelets. In addition to visualizing platelets with mAb, we have utilized the CD41-YFPki/+ mouse [22] to validate our approach of labelling platelets with anti-CD49b. The behaviour of platelets in LPS treated CD41-YFPki/+ mice, a model where no exogenous label is added to the platelets, parallels that observed with anti- CD49b in LPS treated C57BL/6 mice. As a result we have been able to confirm that labelling of platelets with anti-CD49b does not alter their response to an inflammatory stimulus and validates the use of this mAb for the visualization of platelet dynamics in vivo. In CD41-YFPki/+ mice, only a percentage of platelets express YFP and thus are visible by intravital fluorescence microscopy. Whereas it is not possible to measure the size of a given platelet aggregate in this model, it is possible to observe individual platelets within aggregates. This allows for study of individual platelet behaviour and adhesion, even in areas of massive inflammation. Through the use of additional in vivo labels, such as anti-Gr-1 and anti-F4/80, we are able to observe and track the interactions between platelets, neutrophils and Kupffer cells within the LPS challenged liver. Interestingly, Kupffer cells bound the most platelets although the purpose platelet adherence to these cells remains to be determined. Using these two techniques, we have for the first time, observed in vivo platelets under both control, and inflammatory conditions. From these experiments we have identified several novel aspects of platelet behaviour. In some tissues, such as the liver, platelets are observed to frequently interact with the blood vessel walls under basal conditions, briefly touching down and then releasing back into circulation, appearing to probe or patrol the unactivated endothelium. In contrast, other tissues, such as the brain, demonstrate much less frequent platelet-endothelial cell interac- tions under control conditions. Following LPS stimulation, we observe large numbers of platelets interacting with adherent neutrophils, appearing to both roll across the surface of the neutrophil and to firmly adhere to these cells. Platelets are also observed to directly interact with other cell types, such as Kupffer cells and endothelial cells. In contrast to previous reports, few neutrophils are observed being recruited to adherent platelets. Furthermore, following LPS stimulation, platelets are observed to form large, dynamic aggregates that are continually expanding and sloughing off into circulation. These aggregates are dependent on CD18-mediated adhesion and blockade of this integrin completely disrupts the formation of these structures. Finally, in addition to interactions with adherent cells, platelets were also observed to interact with circulating neutrophils following LPS treatment. These circulating platelet-neutrophil aggregates are similar to platelet-leukocyte aggregates (PLA) frequently found in the blood of septic patients [45–48], and are thought to contribute to disease pathogenesis, potentially through the occlusion of organ microvasculature. This work represents the first in vivo observation of these platelet-neutrophil aggregates and future studies will be aimed at understanding the formation and role of these aggregates in severe inflammatory disease. Intravital Visualization of Platelets Through the use of multiple techniques for platelet labelling, we report that anti-CD49b provides an accurate and comprehensive picture of platelet dynamics in a variety of tissues. We were able to measure the number and size of platelet aggregates, to track individual platelet behaviour, and to study the numerous interactions between a given platelet and its surrounding environment. Although anti-CD49b may have some effect on platelet binding to collagen, and as such may not be appropriate for some models of platelet recruitment, the ability to label and visualize platelets in vivo using this antibody, without affecting the ability of platelets to bind important mediators such as fibrinogen or von Willebrand factor, affords many new opportunities for the study of cellular recruitment to sites of inflammation, platelet interactions with bacteria or parasites, platelet-neutrophil-endo- thelium interactions, and the role of platelets in modulating cellular activation, such as the production of NETs by neutrophils [6]. These new experimental possibilities will greatly expand our knowledge of these processes and will help facilitate the development of novel treatment approaches and strategies for numerous conditions involving platelet activation or dysfunction. Methods Ethics Statement 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). Mice 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), unconju- gated 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 mg of anti-CD18 mAb 20 min prior to LPS treatment. Platelet depletion was performed by i.p. injection of 100 ml 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 mg/kg LPS in 200 ml of PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e25109