Figure 1
Figure 1. Intra- and extracellular VWF dots in PMA-stimulated endothelial cells. Human umbilical vein endothelial cells (HUVECs) were secretagogue-challenged for 20 minutes with 80nM phorbol 12-myristate 13-acetate (PMA). (A) Immunolabeling of extracellular von Willebrand Factor (VWF; cells were fixed but not permeabilized) shows VWF dots and VWF strings. The dots, which tend to cluster, appear to give rise to the strings. Scale bar is 2 μm. (B) Schematic drawing of part of a HUVEC labeled postfixation and postpermeabilization for VWF. The elongated Weible-Palade bodies (WPBs; green) that have not been exocytosed during the 20 minutes of stimulation are present in the same area as the VWF dots (blue). Most filaments (red) emanate from the same area as well. Scale bar represents 3 μm. (C) Anti-VWF antibodies present during secretagogue challenge prevented formation of VWF strings but not of VWF dots (green channel, left panel). Following fixation and permeabilization, a second VWF antibody (red channel, middle panel) used to label intracellular VWF revealed additional VWF dots that were not labeled with the extracellular anti-VWF antibodies. The right panel depicts the overlay of the green and red channels. Closed arrowheads indicate VWF dots that were not accessible to the extracellular anti-VWF antibodies, open arrowheads indicate partially accessible VWF dots, and arrows indicate fully accessible VWF dots. Scale bar is 2 μm. (D-E) Close-ups of VWF dots labeled extra- or intracellularly (green and red channels, respectively), showing clusters of dots with WPBs. Note that some VWF dots were only partially accessible to the extracellular label. Scale bar is 1 μm. (F) Double-labeling with anti-VWF antibodies during secretagogue challenge (green channel, left panel) and antipropeptide antibodies after fixation and permeabilization (red channel, middle panel). Some VWF dots were also immunoreactive for the propeptide (closed arrowheads), while not all propeptide dots were accessible to the extracellular VWF antibodies (open arrowheads). Scale bar is 2 μm. (G) Double-labeling with anti-VWF (green channel, left panel) and anti-CD63 (red channel, middle panel) antibodies. CD63 labeled WPBs (open arrowheads) and VWF dots (closed arrowheads). Scale bar is 2 μm. All images were acquired with a Leica SL confocal laser-scanning microscope, using a 63× oil-immersion objective with a numeric aperture of 1.40 (Leica HCX PL APO). Fluorochromes used were FITC (conjugated to sheep anti-VWF antibodies; green channel) and Alexa Fluor 568 (conjugated to secondary goat anti–rabbit or goat anti–mouse antibodies; red channel). Minor adjustments of dynamic range (linear adjustments only) were carried out in Adobe Photoshop.

Intra- and extracellular VWF dots in PMA-stimulated endothelial cells. Human umbilical vein endothelial cells (HUVECs) were secretagogue-challenged for 20 minutes with 80nM phorbol 12-myristate 13-acetate (PMA). (A) Immunolabeling of extracellular von Willebrand Factor (VWF; cells were fixed but not permeabilized) shows VWF dots and VWF strings. The dots, which tend to cluster, appear to give rise to the strings. Scale bar is 2 μm. (B) Schematic drawing of part of a HUVEC labeled postfixation and postpermeabilization for VWF. The elongated Weible-Palade bodies (WPBs; green) that have not been exocytosed during the 20 minutes of stimulation are present in the same area as the VWF dots (blue). Most filaments (red) emanate from the same area as well. Scale bar represents 3 μm. (C) Anti-VWF antibodies present during secretagogue challenge prevented formation of VWF strings but not of VWF dots (green channel, left panel). Following fixation and permeabilization, a second VWF antibody (red channel, middle panel) used to label intracellular VWF revealed additional VWF dots that were not labeled with the extracellular anti-VWF antibodies. The right panel depicts the overlay of the green and red channels. Closed arrowheads indicate VWF dots that were not accessible to the extracellular anti-VWF antibodies, open arrowheads indicate partially accessible VWF dots, and arrows indicate fully accessible VWF dots. Scale bar is 2 μm. (D-E) Close-ups of VWF dots labeled extra- or intracellularly (green and red channels, respectively), showing clusters of dots with WPBs. Note that some VWF dots were only partially accessible to the extracellular label. Scale bar is 1 μm. (F) Double-labeling with anti-VWF antibodies during secretagogue challenge (green channel, left panel) and antipropeptide antibodies after fixation and permeabilization (red channel, middle panel). Some VWF dots were also immunoreactive for the propeptide (closed arrowheads), while not all propeptide dots were accessible to the extracellular VWF antibodies (open arrowheads). Scale bar is 2 μm. (G) Double-labeling with anti-VWF (green channel, left panel) and anti-CD63 (red channel, middle panel) antibodies. CD63 labeled WPBs (open arrowheads) and VWF dots (closed arrowheads). Scale bar is 2 μm. All images were acquired with a Leica SL confocal laser-scanning microscope, using a 63× oil-immersion objective with a numeric aperture of 1.40 (Leica HCX PL APO). Fluorochromes used were FITC (conjugated to sheep anti-VWF antibodies; green channel) and Alexa Fluor 568 (conjugated to secondary goat anti–rabbit or goat anti–mouse antibodies; red channel). Minor adjustments of dynamic range (linear adjustments only) were carried out in Adobe Photoshop.

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