Treat 8 well μ slides

Immunofluorescence studies were performed with transfected HEK293 as previously described [26 (link)]. HEK293 cells were seeded in Ibidi treat 8-well μ-slides (Ibidi, Martinsried, Germany) 24h prior transfection with Lipofectamine LTX (Invitrogen) and VWF-plasmid-constructs in vector pcDNA3 [5 (link)]. Antibodies used were: rabbit anti-VWF (DAKO, Hamburg, Germany, 1:1,000), mouse anti-Protein Disulfide Isomerase (PDI) (Abcam, Cambridge, UK, 1:500), goat anti-rabbit AF488 (Invitrogen, 1:5,000), goat anti-mouse AF 546 (Invitrogen, 1:5,000). Images were captured at RT with a confocal microscope (TCS SP5, Leica, Wetzlar, Germany). For settings, please refer to legend of Fig. 4.

HEK293 cells were cultured in Dulbecco Modified Eagle Medium (DMEM,
Invitrogen) with 10% [v/v] fetal bovine serum (Invitrogen) and 1%
penicillin/streptavidin at 37°C and 5% CO₂. For
immunofluorescence HEK293 cells were seeded in Ibidi treat 8-well
μ-slides (Ibidi, Martinsried, Germany) 24hrs prior
transfection with Lipofectamine LTX (Invitrogen) according to the manufacturers
instructions. 48hrs after transfection cells were washed twice with 1x PBS and
fixed with 3% paraformaldehyde (PFA) in PBS for 10 min at 37°C, then the
cells were permeabilized using 0.3% Triton in PBS for 3min at 37°C and
finally blocked with 1% BSA in PBS for 20min at 37°C. After each step
cells were washed twice with PBS. Antibodies used were: rabbit anti-VWF (DAKO;
1:1,000), mouse anti-PDI (abcam, ab2792, 1:400), goat anti-rabbit AF488
(Invitrogen, 1:5,000), goat anti-mouse AF546 (Invitrogen, 1:5,000). Z-stacks
were recorded at RT with a confocal microscope (TCS SP5, Leica, Wetzlar,
Germany) using an HC PL APO CS2 63.0 x 1.40 OIL UV objective and the following
settings: an image size of 512 x 512 pixels, laser power of the 543 nm and 488
nm lasers was set to 9% and 20%, respectively. 3D reconstruction was performed
using the ImageJ software [59 (link)]. Three
independent experiments were performed.