Correlative light and electron microscopy on forming WPBs in propeptide-EGFP transfected HUVECs. HUVECs transfected with propeptide-EGFP were fixed at several time points after transfection for CLEM. (A) Fluorescence images of HUVECs showing forming WPBs 4, 6, and 8 hours after transfection (hpt) (green channel). The nuclei were stained with DraQ5 (blue channel). The presented images are maximum intensity projections of deconvoluted z-stacks. The boxed WPBs represent 2 examples that were correlated to the corresponding TEM sample as shown in B and C. (B) Overlay of the fluorescence image shown in A and the electron micrograph of a corresponding TEM section. Boxes with asterisks represent 2 examples of WPBs in which the morphology is shown in C. (C) Electron micrographs of correlated WPBs. After 4 hours of transfection, the immature WPBs display a very electron lucent lumen. After 6 and 8 hours of transfection, the WPB lumen becomes more condensed. Also the VWF tubules become more visible within the lumen of the WPB. Scale bars: (A-B) 5 µm; (C) 250 nm. Fluorescence images were acquired using a Leica SP5 confocal with a ×63 oil immersion objective with a numerical aperture of 1.4. Fluorochromes used are EGFP tagged to the propeptide of VWF (propeptide-EGFP, green) and DraQ5 (blue). Deconvolution of the confocal images was performed using Huygens Pro. Electron micrographs were acquired using an FEI Tecnai 12 at 120 kV and using an FEI Eagle 4k × 4k CCD camera. Overlays as shown in B were prepared in Adobe Photoshop CS6.