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. 2003 Jun 2;197(11):1585–1598. doi: 10.1084/jem.20021868

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Copyright © 2003, The Rockefeller University Press

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Figure 2. — Tissue factor accumulation in the developing arterial thrombus. (A) Left: characterization of rabbit anti–mTF (152–166) antibodies. Western blot of tissue factor with affinity-purified rabbit anti–mTF (152–166) antibodies. STO cells, red cells, and platelets were lysed and the proteins in the lysate were separated on a 10% SDS-PAGE gel under reducing conditions. Lanes 1 and 5, 5 μg STO cell lysate; lanes 2 and 6, 7 μg STO cell lysate; lane 3, 5 μg murine red cell lysate; lane 4, 7 μg murine red cell lysate. Lanes 1–4 were developed with anti–mouse tissue factor antibody and lanes 5 and 6 were developed with an irrelevant antibody, anti-CD41. Arrow indicates molecular weight of tissue factor. Middle: comparison of tissue factor fluorescence detected with rabbit anti–mTF (152–166) antibodies (bar 2) or nonimmune IgG control (bar 1). The ratio of the integrated tissue factor fluorescence (F_TF) to the integrated platelet fluorescence (F_P) in thrombi generated in wild-type mice is shown based upon multiple independent experiments. P < 0.0042. Right: image of tissue factor in a thrombus detected using rabbit anti–mTF (152–166) antibodies. Top: Thrombus detected with anti-mTF (152–166) antibodies and anti-CD41 antibody directed against platelets. Bottom: Thrombus imaged with nonimmune IgG and anti-CD41 antibody directed against platelets. Red, platelets; green, tissue factor; yellow, platelets plus tissue factor. (B) Intravital images of arterial thrombi. Tissue factor was detected using Alexa 488–conjugated anti-tissue factor antibodies infused into the systemic circulation. The fluorescence image, recorded digitally, is presented as the green pseudocolor. Brightfield images of the thrombi are indicated by arrowheads. Images are representative of 12 thrombi formed in 2 arterioles in 2 mice of each genotype. (C) Time course of tissue factor antigen accumulation in the developing arterial thrombus in wild-type and genetically altered mice. Tissue factor was detected using sheep anti–tissue factor antibodies infused into the systemic circulation followed by Alexa 660–conjugated donkey anti–sheep IgG. Sheep IgG was substituted for anti-tissue factor antibody, control. Each curve represents the raw digital data of a single representative experiment. WT, wild-type mouse; PSGL^−/−, PSGL-1 null mouse; P-selectin^−/−, P-selectin null mouse.