Figure 4. Characterization of platelet binding in the livers of mice treated with intravenous LPS using CD41-YFPki/+ mice. Representative fields of view of livers from untreated (A) and LPS (B) treated CD41-YFPki/+ mice. Visualization of YFP+ platelets and liver autofluorescence (Ai, Bi), neutrophils labelled with Alexa Fluor 647- conjugated anti-Gr-1 (Aii, Bii), Kupffer cells labelled with PE-conjugated anti-F4/80 (Aiii, Biii), and overlay of fluorescent channels (Aiv, Biv). Intravital Visualization of Platelets Examples of platelets interacting with the liver sinusoid away from neutrophils or Kupffer cells (long white arrows), platelets interacting with neutrophils (short white arrows), platelets interacting with Kupffer cells (short yellow arrows), and platelets appearing to interact with both Kupffer cells and neutrophils (long yellow arrows). All scale bars, 20 mm. (C) Quantification of the number of YFP+ platelets bound to neutrophils, Kupffer cells and liver sinusoids per field of view within livers of untreated and LPS treated CD41-YFPki/+ mice. *** p,0.001, ** p,0.01. doi:10.1371/journal.pone.0025109.g004 conclude that labelling with anti-CD49b neither blocks, nor enhances platelet recruitment to the liver in response to LPS challenge and allows for the observation of normal platelet behaviour in vivo. Visualization of platelet recruitment in other tissues using anti-CD49b We have demonstrated the novel use of PE-conjugated anti- CD49b to readily visualize platelet dynamics in the mouse liver; however, this technique can also be applied to other tissues. Intravital visualization of the mouse brain microvasculature using anti-PECAM-1 reveals no platelet aggregation, and rare platelet- endothelial interactions under resting conditions (Fig. 5A, Movie S12). In contrast to untreated mice, neutrophil and platelet recruitment can be readily observed in the brain 4 h following LPS treatment (Fig. 5B, Movie S13). Platelets can be seen adhering to primarily to adherent neutrophils with a few interactions with the vascular endothelium. Almost all of these interactions occur in the postcapillary venules of the brain with little recruitment seen in brain arterioles. Similar to what was observed in the liver, these platelet interactions appear to be very dynamic, with platelets adhering to neutrophils or endothelium and forming unstable aggregates that, after a period of time, slough off into the circulation. Platelets can also be observed in muscle microvasculature using PE-conjugated anti-CD49b. Intravital visualization of postcapil- lary venules in the cremaster muscle using anti-PECAM-1 reveals little platelet adherence in untreated animals, although some platelets can be seen interacting with the vascular endothelium, appearing to touch-down and then quickly release (Fig. 6A, Movie S14). Following LPS exposure, however, dramatic neutrophil and platelet recruitment can be observed (Fig. 6B, Movie S15). Again, platelets are seen to interact with, and adhere to, both the vessel endothelium and neutrophils in a dynamic fashion. These videos also demonstrate that platelet interaction with neutrophils does not require the neutrophil to be adherent. In some instances, neutrophils covered in platelets can be observed in circulation and seen rolling on the cremaster vascular endothelium. Discussion Platelets are emerging as central players in both innate and adaptive immunity [1,3,4,6]. As such, a more in depth knowledge of their role in inflammation, immune cell recruitment and cellular activation is critical to our understanding of these processes. Intravital microscopy can reveal elegant details about the dynamic interactions between platelets and their surrounding environment, information that is not possible to obtain from other techniques. Although recent advances have been made in the ability to label and visualize platelets in the living animal, isolation of platelets for labelling or injection of mAb against specific platelet receptors such as CD41 have a number of limitations. PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e25109