Fig. 6. Translocation of PrP^C to intercellular junctions is dependent on PrP^C expression by adjacent cells. Double immunofluorescence labeling of PrP^C and ß-catenin in a mixed primary culture of wild-type (WT) and PrP^C-deficient knockout (KO) mouse brain endothelial cells. Cells were permeabilized with 0.05% saponin for 1 hour before incubation with anti-PrP^C (SAF32, 2 µg/ml) monoclonal antibody plus anti-ß-catenin rabbit polyclonal antibodies (1 µg/ml) for 16 hours at 4°C. ß-catenin and PrP^C were revealed using Cy2-conjugated anti-rabbit antibodies (A) and Cy3-conjugated anti-mouse antibodies (B), respectively. Images were collected with a confocal fluorescence microscope. Arrows indicate a junction between two WT cells showing a positive staining for ß-catenin (A) and PrP^C (B), arrowheads indicate a junction between one WT cell and one KO cell showing a positive staining for ß-catenin (A) but no staining for PrP^C (B). A merged image of the same field is presented in the right hand panel (merged), with PrP^C staining in red and ß-catenin staining in green: co-localization (yellow) of PrP^C and ß-catenin is observed only at junctions between two adjacent WT cells. Bar, 20 µm.