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Figure 6 The association of FITC-fibrinogen with quiescent platelets in blood anticoagulated with CTI (32 µg/ml) and exposed to FITC-fibrinogen (300 mg/dl) for 15 min (micrographs on left: A, C, and E). The platelets were fixed in Optilyse-C and exposed subsequently to 0.1% Triton X-100 and anti-P-selectin IgG. After washing, platelets were exposed to a secondary Alexa-conjugated anti-mouse IgG. The concentration of fibrinogen in blood from this subject was 360 mg/dl. The total concentration of fibrinogen (endogenous plus FITC-fibrinogen) to which the platelets were exposed was 330 mg/dl. The micrographs on the right (B, D, and F) show results with blood drawn into CTI spiked with ReoPro (10 µg/ml). For each set of observations, the micrograph on the top (A and B) is a representative platelet imaged with phase contrast with the transmitted light detector of the confocal microscope merged with fluorescence (excitation 488 nm) demonstrating FITC-fibrinogen (green signal). The micrograph in the center (C and D) is the same platelet visualized with phase-contrast transmitted light and fluorescence (excitation 568 nm) demonstrating P-selectin expression (red signal). The red signal localizes -granules. The micrograph on the bottom shows the same platelet viewed by multiplying fluorescent images (excitation 488 and 568) and thus the degree of co-localization of fibrinogen-FITC and P-selectin (yellow color). Fluorescence in images A and C are multiplied to create E, and fluorescence in images B and D are multiplied to create F. Yellow demonstrates localization of fibrinogen in -granules. Bar = 5 µm; all images are same magnification.