Immunofluorescence microscopy
Transfected HEK293 cells were fixed and stained as described previously
. Primary antibodies anti-VWF (Abcam, England) and anti-protein
disulfide isomerase (PDI; Life technologies, USA) were used to visualize
intracellular VWF and endoplasmic reticulum (ER), respectively. Z-stack
images were acquired with Zeiss Apotome.2 microscopy (Carl Zeiss,
Germany). The quantitative co-localization investigation was done using
Zeiss ZEN 2 (blue edition; Carl Zeiss, Germany). Analysis for each set
of comparisons was performed on z-series of images of a minimum of 100
cells from two independent experiments (each in triplicate). The
comparative degree of co-localization was calculated as mean Pearson’s
correlation coefficients.