Intravital Visualization of Platelets Figure 5. Intravital visualization of platelets labelled with PE- conjugated anti-CD49b within the mouse brain. Spinning-disk confocal-microscopic imaging of brain pial arterioles and postcapillary venules in untreated (A) and LPS treated mice (B). Visualization of endothelium with Alexa Fluor 488-conjugated anti-CD31 (Ai, Bi), neutrophils labelled with Alexa Fluor 647-conjugated anti-Gr-1 (Aii, Bii), platelets stained with PE-conjugated anti-CD49b (Aiii, Biii), and multi-channel overlay (Aiv, Biv). There was a marked absence of platelet or neutrophil adhesion in brain pial vessels of untreated mice (A), however, profound neutrophil and platelet recruitment within brain venules (faint CD31 staining), but not in brain pial arterioles (bright CD31 staining), following LPS treatment (B). Small arrows identify platelets interacting with neutrophils; large arrows identify platelets interacting with the endothelium. All scale bars, 20 mm. doi:10.1371/journal.pone.0025109.g005 Figure 6. Intravital visualization of platelets labelled with PE- conjugated anti-CD49b within the mouse cremaster muscle. Spinning-disk confocal-microscopic imaging of cremaster postcapillary venules in untreated (A) and LPS treated mice (B). Visualization of endothelium with Alexa Fluor 488-conjugated anti-CD31 (Ai, Bi), neutrophils labelled with Alexa Fluor 647-conjugated anti-Gr-1 (Aii, Bii), platelets stained with PE-conjugated anti-CD49b (Aiii, Biii), and multi-channel overlay (Aiv, Biv). A small number of platelets are seen to interact with the postcapillary endothelium in the cremaster muscle under basal conditions (A). Neutrophil recruitment and platelet interactions increase dramatically in the vessels of the cremaster muscle following LPS treatment (B). Small arrows identify platelets interacting with neutrophils; large arrows identify platelets interacting with the endothelium. All scale bars, 20 mm. doi:10.1371/journal.pone.0025109.g006 Intravenous administration of anti-CD49b rapidly labels the total peripheral platelet population and can facilitate the visualization of platelets by intravital microscopy. The use of the anti-CD49b mAb HMa2 for the labelling of platelets has several advantages over previous approaches. First, i.v. administration of anti-CD49b, unlike CD41 [14], does not induce an adverse systemic response. Second, HMa2 is widely available and can be ordered pre-conjugated to a number of different fluorophores. Third, this technique can be used to visualize platelets in mice with various genetic backgrounds and gene knockouts whereas models PLoS ONE | www.plosone.org 7 September 2011 | Volume 6 | Issue 9 | e25109