Formation of WPBs in HUVECs after PMA stimulation. HUVECs stimulated with PMA were washed with medium and were left to recover for 2 to 8 hours. (A) Immuno-fluorescent labeling for VWF (VWF-FITC, green channel), the trans-Golgi network (TGN 46 Alexa 568, red channel), and the nucleus (4′,6-diamidino-2-phenylindole, dihydrochloride, blue channel) after 2, 4, 6, and 8 hours of recovery. We found that the cells were devoid of WPBs after 2 hours of recovery. We followed the production of new WPBs from that time point onward. (B-G) VWF-specific immunogold labeling on TEM samples prepared after 2, 4, 6, and 8 hours of recovery. Gold particles are 15 nm in size. (B-C) Immuno-gold labeled structures found after 2 hours of recovery. (B) Large vesicle found close to the cell membrane that contains VWF specific immunogold label. (C) Lysosome-like structure showing VWF specific immunolabeling. (D-Di) Immature WPBs positively labeled for VWF near the Golgi apparatus (Go) that was found in TEM samples prepared after 4 hours of recovery. (E-Ei) A more advanced immature WPB positively labeled for VWF that was found close to the Golgi (Go) in TEM samples prepared after 6 hours of recovery. In this WPB, longer VWF tubules are observed compared with the 4-hour condition. (F) Condensed mature WPBs found after 8 hours of recovery. (G) VWF-containing vesicle found after 8 hours of recovery that appears to contain disorganized VWF tubules. Scale bars: (A) 5 µm; (B-G) 250 nm. Fluorescence images were acquired using a Leica SP5 or Leica SP8 confocal with a ×63 oil immersion objective with a numerical aperture of 1.4. Fluorochromes used are FITC (conjugated to sheep anti-VWF antibodies, green), Alexa 568 (conjugated to goat anti-rabbit antibodies, red), and DraQ5 (blue). Electron micrographs were acquired using an FEI Tecnai 12 at 120 kV and using an FEI Eagle 4k × 4k CCD camera.