Intracellular distribution of recombinant VWF in late endosomal compartment. Cells transfected with vectors and siRNA indicated above the images (A-H) were coimmunostained for VWF (tetramethylrhodamine isothiocyanate, red) and mannose 6-phosphate receptor (fluorescein isothiocyanate, green) as late endosome marker. Images were taken with Carl Zeiss LSM 510 equipped with a Fluar 40×/1.3 oil immersion objective at room temperature on fixed cells mounting in glycerol/1,4 diazabicyclo[2.2.2]octane/4,6-diamidino-2-phenylindole medium. Briefly, 488- and 543-nm excitation wavelengths were provided, respectively, by Argon/2 and HeNE laser sources at a 5% intensity. Superimposition of red and green fluorescence, not specifically caused by colocalization, was excluded by applying to the green channel a beam path admitting the acquisition of fluorescence composed among 505 and 550 nm and to the red channel a light path excluding all fluorescence less than 560 nm. Pinhole values ranged from 40 to 80 μm. Images in the top panels were digitally zoomed in twice the original size with LSM examiner software Version 3.0 (Carl Zeiss).