Exposure of activation and secretion marker LAMP-1 in comparison with platelet accumulation in vivo after laser injury. Anti–mouse CD41 Fab fragments conjugated to Alexa 647 and anti–mouse LAMP-1 antibodies conjugated to Alexa 488 were infused to label platelets and LAMP-1, respectively. Injuries were induced by laser pulses to cremaster arteriole vessel walls and subsequent LAMP-1 accumulation and thrombus formation recorded over time. (A) Images from a representative experiment showing fluorescence over time overlaying brightfield data before and after vessel injury. LAMP-1 accumulated rapidly at the vessel wall (green) followed by platelet accumulation (red) or presence of both signals (yellow). The fluorescence signal is shown binarized for ease of visual interpretation. (B) Kinetic curves displaying the median integrated platelet fluorescence (black curve on right y-axis) and median integrated LAMP-1 fluorescence (gray curve on left y-axis) for 18 thrombi in 3 wild-type mice. LAMP-1 fluorescence originating from platelets and not the endothelium was eliminated by subtracting any LAMP-1 fluorescence in pixels where Alexa 647 fluorescence was observed.