Intravital Visualization of Platelets Figure 2. Quantification of platelet aggregation in the livers of mice treated with intravenous LPS using PE-conjugated anti- CD49b. Intravital microscopy of livers from untreated mice (Ai–Aiv), LPS treated mice (Bi–Biv) and mice pre-treated with an anti-CD18 blocking mAb followed by LPS (Ci–Civ). Representative fields of view at low power (106objective) magnification (Ai, Bi, Ci) illustrating platelet aggregation in response to LPS and inhibition of aggregation following pre-treatment with an anti-CD18 blocking Ab. Platelets labelled with PE- conjugated anti-CD49b (red); neutrophils labelled with Alexa Fluor 647- conjugated anti-Gr-1 (blue). Arrows denote large platelet aggregates. Higher power (206 objective) extended focus images (Aii, Bii, Cii) and 3D opacity models (Aiii, Biii, Ciii) rendered from 29–30 z planes illustrating the extent of platelet aggregation in LPS treated mice. All scale bars, 20 mm; grid 25.7 mm. Select models of neutrophil-platelet aggregates (Aiv, Biv, Civ) extracted (as indicated by the white arrows) and enlarged from panel (iii). (Di, Dii) Quantification of the number of platelet aggregates equal to, or larger than the indicated sizes. *** p,0.001, * p,0.05, n.s. not significant (E) Time lapse images of neutrophil-platelet interactions illustrating the dynamic nature of the platelet aggregates within the liver. Long white arrow identifies a large platelet aggregate tearing off of an adherent neutrophil, short yellow arrow indicates direction of blood flow within the sinusoid. Sequential images represent 2.3 s intervals. Scale bar, 20 mm. doi:10.1371/journal.pone.0025109.g002 In support of this model, depletion of either neutrophils or Kupffer cells dramatically inhibits platelet aggregation, indicating these cells are essential to the process (Fig. S5). Therefore, it is likely that circulating platelets must touch-down on, and anchor to, a neutrophil or Kupffer cell as a first step towards the generation of larger platelet aggregates. Through the use of intravital microscopy we are able to visualize the behaviour of platelets within the liver sinusoids and their interactions with adherent neutrophils. It is clear from these studies that the platelet aggregates that form in response to LPS are not static entities, but rather, are dynamic structures, continually expanding and sloughing off (Fig. 2E, Movie S7 (white arrow identifies a large platelet aggregate tearing off of a neutrophil)). These platelet aggregates often appear to ‘‘flow’’ across the surface of an adherent neutrophil (Movie S6, S7). In addition to platelet aggregates associated with adherent neutro- phils, we are also able to observe circulating platelet-neutrophils aggregates (Fig S6, Movie S8). Neutrophils, covered with large platelet aggregates can be seen flowing through the liver sinusoids, with little interaction between the neutrophil and the vessel walls. These observations would not be possible with other techniques such as staining of tissue sections, intravital observation of adoptively transferred platelets, or in vivo labelling of platelets with rhodamine 6-G and are essential to our understanding of platelets in the host immune response. Use of CD41-YFPki/+ mice for the intravital study of platelet recruitment to the mouse liver The generation and characterization of CD41-YFPki/+ mice has been previously reported [22], although, to date these mice have not been used for the intravital study of platelet behaviour. In these mice, a percentage of platelets express YFP (Fig. S7) and thus are visible using fluorescence microscopy without the need to add an exogenous label. As such, these mice can be used to confirm that the platelet adhesion observed in Fig. 2 is independent of the labelling of platelets by anti-CD49b. As the percentage of YFP-labelled platelets varies significantly from mouse to mouse (from less than 10% to more than 40%) [22], all animals must be pre-screened by flow cytometry to ensure individuals in the control and treated groups have comparable PLoS ONE | www.plosone.org 4 September 2011 | Volume 6 | Issue 9 | e25109