endothelial interactions under basal conditions are independent of surgical manipulation of the tissue. To further establish the identity of these particles as platelets, mice were pretreated with anti-thrombocyte serum for 20 h prior to intravital microscopy. In these platelet-depleted animals there was an absence of labelled particles following injection of PE- conjugated anti-CD49b (Fig. 1Ci, Cii, Movie S4). The lack of labelling in thrombocyte depleted mice, together with the flow cytometric analysis of CD41 and CD49b co-staining of mouse peripheral blood, positively identifies the labelled particles observed by intravital microscopy as platelets. Mice receiving intravenous anti-CD49b do not present any of the adverse health effects observed using anti-CD41 antibodies [14] and have remained stable for more than three hours, allowing for the intravital observation of platelets throughout this timeframe (unpublished observations). It should be noted that this staining also labels NK cells within the tissue and blood (Fig. S2). These cells are extremely rare, fewer than 10/low power field of view in the mouse liver, despite this tissue being a main target and resident site for NK cells. They are much larger than a platelet, and their staining is fainter than that of the platelets, allowing for direct observation of platelet-NK cell interactions if needed. As such, these cells do not interfere with intravital platelet analysis. No iNKT cells were labelled with this approach. Treatment of Cxcr6gfp/+ mice, whose iNKT cells are GFP-positive, failed to result in dual labelling of these cells (data not shown). Use of anti-CD49b for the intravital study of platelet recruitment to the mouse liver Treatment of mice with LPS has been reported to induce the recruitment of neutrophils and platelets to various tissues [6]. In sharp contrast to the liver of control animals (Fig. 2Ai–iv), intravital analysis of livers from mice treated with 1 mg/kg LPS i.v. for 4 h clearly demonstrates profound neutrophil recruitment (Fig. 2Bi, Bii). Furthermore, analysis of peripheral blood in these mice reveals a drop in the circulating platelet count (Fig. S3). Intravenous administration of PE-conjugated anti-CD49b allows, for the first time, the direct intravital visualization of endogenous platelet recruitment to this tissue. Platelets are readily observed to be interacting primarily with the large number of neutrophils already adherent within the liver. Many fewer interactions are seen between platelets and the liver sinusoids and almost no interaction between circulating neutrophils and adherent platelets can be observed. Many of the recruited platelets form aggregates, often the same size as, or larger than, the adherent neutrophils (Fig. 2Bi, Movie S5). Extended focus images generated from stacks of multiple xy planes at higher magnification reveals many of these aggregates are associated with neutrophils (Fig. 2Bii), forming from an initial adherence of platelets to a neutrophil followed by platelet-platelet interactions facilitating the expansion of the growing aggregate. Often, aggregates are seen to be spanning between two proximal neutrophils and appear to greatly reduce the blood flow in a number of liver sinusoids. By rendering 3D models of these z stacks with the Volocity software package we are able to visualize these platelet-neutrophil interactions from a number of different perspectives (Fig. 2Biii, Movie S6). Closer inspection of representative neutrophils from LPS treated mice illustrates the interaction between the large platelet aggregates and the adherent neutrophils (Fig. 2 Biv). These platelet aggregates cover large portions of the neutrophil surface and appear to ‘‘wrap around’’ the neutrophil. Through the generation of these z stacks, and the subsequent 3D images, we are able to determine these platelet aggregates are directly associated with the adherent Intravital Visualization of Platelets neutrophils, an observation that would not be possible through the imaging of individual static sections. These findings are consistent with the observation that neutropenia precedes thrombocytopenia in this model of inflammation [2]. Unlike previous reports using other models of inflammation, we rarely see platelets first binding to endothelium followed by neutrophil recruitment to these adherent platelets [27–29]. In contrast, the few adherent neutrophils present in the liver of untreated mice are largely free of platelet interactions (Fig. 2Aiii, Aiv (representative 3D models)). In the few examples of neutrophil- platelet interactions in untreated mice, we do not see substantial platelet-platelet interactions and as such there is a failure to generate larger platelet aggregates. It is important to note our in vivo platelet labelling excludes platelet activation due to platelet isolation or in vitro labelling procedures. Visualization of platelets within the mouse liver in response to LPS treatment allows for the characterization of receptors and adhesion molecules involved in platelet recruitment. Past reports have implicated the adhesion molecule CD18 in the binding of platelets to neutrophils [18,30–32], dendritic cells [33], and T cells [34], although to date, the effect of CD18 blockade on platelet recruitment has not been visualized in vivo. As previously reported by our group [35,36], pre-treatment of mice with a blocking Ab directed against CD18 has no effect on neutrophil recruitment to the liver in response to LPS (Fig. 2Ci). By contrast, CD18 blockade had a dramatic effect on platelet recruitment to the inflamed liver (Fig. 2Ci–Civ). Although individual platelets appeared to be recruited independent of CD18, large aggregates failed to form in mice treated with anti-CD18 blocking antibody. Furthermore, because i.v. administration of anti-CD49b labels all platelets, it is possible to measure the size of the numerous platelet aggregates. The area of staining corresponding to each platelet aggregate was measured and the number of aggregates equal to, or greater than, specific size thresholds were enumerated. We observed a significant increase in both the total number of recruited platelets and the size of platelet aggregates within the livers of LPS treated mice (Fig. 2Di, Dii). This platelet recruitment and aggregation was significantly inhibited by the pre-treatment of mice with a blocking Ab against CD18. The ability of CD18 blockade to inhibit platelet aggregation is particularly interesting given the fact that platelets have not been reported to express this molecule. One possible mechanism to explain this observation might involve the formation of micropar- ticles. LPS treatment has been reported to result in the generation of neutrophil, endothelial and platelet derived microparticles. These microparticles are formed from the plasma membrane and can express cell surface markers and adhesion molecules derived from the cell from which they were released [37–39]. These particles have been reported to express CD18 and might facilitate platelet aggregation by bridging between ligands on individual platelets. Flow cytometric analysis of platelets from untreated and LPS-treated mice failed to detect any association between platelets and either neutrophil (GR1+) or Kupffer cell (F4/80+) derived membranes (Fig. S4A, B). Furthermore, expression of CD18 was not detected on platelets obtained from untreated or LPS-treated mice, further ruling out the association of platelets with CD18+ microparticles (Fig. S4C). As we were unable to detect CD18 expression by platelets, it is most likely this molecule is expressed by other cells involved in platelet aggregation. In fact, CD18 is highly expressed by both neutrophils and Kupffer cells [40–43], and platelets express a number of ligands for b2 integrins (ICAM-2, Gp1ba, JAM-C) [30,33]. These observations suggest CD18 expressing leukocytes within the liver vasculature bind platelets and initiate aggregation. PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e25109