Colocalization uPAR/caveolin-1 and caveolar rafts analysis by immunofluorescence. Immunostaining of uPAR and caveolin-1 in ECFCs in different experimental conditions. Panels on the left represent uPAR staining (red represents Cy3) using anti-uPAR R3 antibody, which identifies full-length uPAR; central panels correspond to caveolin-1 staining (green represents FITC), using anti-caveolin-1 antibody and right panels show the double staining of ECFCs with anti-uPAR (red) and anti–caveolin-1 (green). Nuclear staining is obtained by the use of 4,6-diamidino-2-phenylindole (blue). Cells were observed with an inverted confocal Nikon Eclipse TE2000 microscope equipped with a Nikon S Fluor 60× oil immersion lens (Nikon). Images were acquired at room temperature, using a standard 1024- by 1024-pixel image format and adjusting the zoom level to match the voxel size to the Nyquist criterion. The pinhole size was always set at 1 airy unit (airy disk), and each plane was Kalman averaged to reduce noise. In each experiment, the same instrumental settings were used for all image acquisitions. All images were Gaussian filtered to eliminate single-pixel noise before analysis (original magnification × 600).