Correlative fluorescence and transmission electron microscopy of VWF dots. HUVECs were stimulated for 20 minutes with 80nM PMA in the presence of FITC-conjugated anti-VWF antibodies to detect extracellularly accessible VWF. (A-A‴) Examples of VWF dots correlated with electron-dense, round-shaped structures outside the cell. (A) Fluorescence micrograph of a HUVEC showing VWF immunofluorescence in green and DAPI in blue. Scale bar represents 10 μm. (A′) Electron micrograph of the cell shown in panel A. (A″) Overlay of the immunofluorescence and electron microscopic images shown in panels A and A′. (A‴) Higher magnification TEM view of the VWF dots in the boxed area in A″. Scale bar represents 1 μm. (B-B″) Example of a cluster of VWF dots correlated with electron-dense, round-shaped structures inside the cell. (B) Fluorescence micrograph of a HUVEC showing VWF immunofluorescence in green and DAPI in blue. Scale bar represents 10 μm. (B′) Electron micrograph of the cell shown in panel B. (B″) Overlay of the immunofluorescence and electron microscopic images shown in panels B and B′. (C-C′) Electron micrographs of 2 consecutive sections corresponding to the boxed area in panel B″, revealing that the cluster of electron-dense VWF dots (asterisks) is contained within a vesicular structure (secretory pod, SP). Scale bar represents 500 nm. Fluorescence images were acquired with a Leica TCS SP5 confocal laser-scanning microscope and a 63× oil-immersion objective with a numeric aperture of 1.4 (Leica HCX Plan APO). Electron micrographs were acquired with an FEI Tecnai 12 TEM at 120 kV and using an FEI Eagle 4kx4k CCD camera.