Fibrin localization partially overlaps with α-granule release in time and space. (A) Photomicrographs showing representative 2-dimensional confocal images illustrating localization of P-selectin and fibrin in a hemostatic plug following laser-induced injury. The image on the left shows the anti-fibrin (green) alone on the brightfield background. The image on the right shows platelets (CD41, red), P-selectin (blue), and fibrin (green) on the brightfield background; see color key at right for overlapping fluorophore colors. All 3 fluorescent channels are shown as binary images. Fibrin was imaged using an anti-fibrin antibody. (B) Representative photomicrographs illustrating localization of fibrin in the extravascular region adjacent to the site of laser injury. The image on the left shows Alexa-488–labeled fibrinogen on the brightfield background. The image on the right shows platelets (CD41, red), P-selectin (blue), and Alexa-488 fibrinogen (green) on the brightfield background. CD41 and P-selectin channels are displayed as binary images. The γ setting of the fibrinogen channel was set to 0.5 to increase the brightness of the extravascular fibrin network. Fibrin(ogen) (green) is observed throughout the vessel lumen and hemostatic plug, but discernible fibrin fibers are only observed in the extravascular space. Bars represent 10 µm. Time-lapse videos of the formation and 3-dimensional reconstructions of the hemostatic plugs in both A and B are provided in supplemental Video 2.