specific nature it also results in the fluorescent labelling of all blood cells, including neutrophils and monocytes, key targets for platelet interactions. This makes it impossible to distinguish aggregates of platelets from leukocytes at sites of inflammation or to visualize platelets directly associated with rhodamine 6-G labelled leukocytes. In contrast, ex vivo staining of purified platelets results in specific labelling and allows for the study of platelet interactions with other cells, however, only a small percentage of the platelets in the recipient animal are labelled following adoptive transfer (endog- enous platelets remain unlabelled). This limits the ability to measure total platelet recruitment, aggregate size or to visualize platelet-platelet interactions. Additionally, platelets are easily activated by shear stress and temperature fluctuation [12,13]. Thus one has to pay particular attention to the platelet isolation, labelling and transfusion to ensure that the adoptively transferred platelets are not activated and that their behaviour upon transfer mirrors that of endogenous platelets. Not trivial is the fact that such experiments require the killing of substantial numbers of additional animals in order to obtain platelets for transfer. More recent approaches have focused on monoclonal antibody (mAb) labelling of platelets in vivo. Early attempts to use mAb against the pan-platelet marker CD41 were not optimal [14]. Although this approach allowed for the visualization of platelets in vivo [15,16], mAb directed against CD41 have been reported to inhibit more than 80% of cellular adhesion to fibrinogen [17], an important molecule in platelet and cellular recruitment to sites of inflammation, and platelet aggregation [14,18]. Other experi- ments have used an mAb directed against the platelet surface receptor GPIbb [19], and although this mAb is able to label platelets in vivo, it blocks the ability of platelets to bind von Willebrand factor [20] and possibly Mac-1, important mediators of platelet adhesion and neutrophil-platelet interactions [21]. The undesired effects of anti-CD41 and anti-GPIbb antibodies make these approaches to platelet labelling less than ideal for the study of platelet responses to inflammatory stimuli. In our efforts to find a better surface marker to facilitate platelet labelling, without blocking platelet function, we have identified CD49b as a suitable target molecule. Fluorescently conjugated antibodies against this marker readily label platelets in vivo with no observable effect on platelet behaviour. Additionally, we have utilized a transgenic mouse expressing yellow fluorescent protein (YFP) under the control of the CD41 promoter [22] to further characterize platelet dynamics and to validate the results obtained by in vivo Ab labelling by CD49b. Finally, we highlight examples of how these approaches, combined with spinning-disk confocal microscopy and 3D reconstruction capabilities, reveal new and important data about platelet function in vivo. Results In vivo labelling of mouse platelets with anti-CD49b In blood, CD49b is expressed on NK cells, a subset of NKT cells and on platelets [23–25]. Due to this restricted expression, we decided to test the suitability of this molecule as a marker for platelets in vivo. Flow cytometric analysis of blood platelets demonstrates the mAb HMa2 directed against CD49b stains nearly all platelets as identified by the pan-platelet marker CD41 (Fig. 1A). This staining appears to be bright, stable and uniform, with a single population of CD49b+ platelets. These results identify this marker as a potential candidate for in vivo labelling of platelets for intravital microscopy applications. Intravenous (i.v.) administration of 1.6 mg of PE-conjugated anti-CD49b rapidly labelled a large number of particles within the liver vasculature that were several fold smaller than leukocytes (Fig. 1Bi, Movie Intravital Visualization of Platelets Figure 1. In vivo labelling of mouse platelets by intravenous injection of the anti-CD49b antibody HMa2. Anti-CD49b uni- formly labels all CD41+ platelets in mouse blood (A). Intravenous injection of PE-conjugated anti-CD49b (red) labels circulating platelets within the mouse liver (Bi –106 objective, Bii –206 objective) and pre- treatment of mice with anti-thrombocyte serum (Ci –106 objective, Cii –206 objective) results in the loss of CD49b+ particles confirming particles labelled by CD49b are indeed platelets. Neutrophils labelled with Alexa Fluor 647-conjugated anti-Gr-1 (blue). All scale bars, 20 mm. doi:10.1371/journal.pone.0025109.g001 S1). These particles were measured to have a diameter of 2.8060.37 mm, which fall into the expected size range of mouse platelets [26]. Unexpectedly, many of these particles were observed to briefly interact with the sinusoid wall or with the few neutrophils present in an untreated liver. Although brief interactions could be observed, these particles almost never remained adherent but rather appear to freely circulate through the liver sinusoids (Fig. 1Bii, Movie S2). To ensure the particle-endothelial interactions observed in control mice were not the result of surgical manipulation of the tissue, we examined particle behaviour within the ear microvas- culature, a tissue that requires no surgical preparation prior to visualization of the blood vessels. Once again, no adherent, and a few interacting particles are seen under resting conditions (Fig. S1, Movie S3). These results indicate that the observed particle- PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e25109