Localization of ADAMTS13 in human endothelial cells by fluorescent microscopy. HUVECs (A,D,G,J), HAECs (B,E,H,K), and ECV304 (C,F,I,L) grown on chamber slides or cryosections of human umbilical veins (M-N) were fixed with ethanol–acetic acid (9:1). The cells or slides were incubated with rabbit anti–ADAMTS13 IgG (20 μg/mL) and mouse anti–VWF IgG (10 μg/mL), followed by incubation of the cells with Cy3-conjugated anti–rabbit IgG and Cy2-conjugated anti–mouse IgG (1:100). The nuclei were stained with 4′, 6′-diamidino-2-phenylindole (DAPI) in the mounting medium. The digital images (40×) were taken under a Nikon inverse fluorescent microscope. ADAMTS13 staining in red is shown in panels A-C. VWF staining in green is shown in panels D-F. Overlapping images for ADAMTS13, VWF, and DAPI staining were shown in panels J-L and N. The yellow staining (white arrows) indicates the areas of ADAMTS13 colocalized with VWF. Cells (not shown) or tissue sections (M) incubated with only secondary antibody were negative.