Colocalization of GPIb subunits and 14-3-3ζ in HEL cells (A to E) and platelets (F to I). Permeabilized platelets and HEL cells were colabeled with antibody pairs. HEL cells were labeled with either (A) anti-GPIbα/FITC (green) and anti-CD62P/CY-3 (red) (negative control), (B) anti–14-3-3ζ/FITC and anti-CD62P/CY-3 (negative control), (C) anti-GPIbα/FITC and anti-GPIX/CY-3 (positive control), (D) anti-GPIbβ/CY-3 and anti–14-3-3ζ/FITC, or (E) anti-GPIbα/CY-3 and anti–14-3-3ζ/FITC. Inset: detector 1, FITC fluorescence intensity; detector 2, CY-3 fluorescence intensity. Yellow fluorescence suggests that the 2 antibodies colocalize to within approximately 200 nm. A and C to E field size, 36 × 36 μm; B field size, 54 × 54 μm. Representative platelet pixel histograms for each antibody pair are shown.

(F to I) and reflect colocalization of GPIX/Ibα (G), GPIbβ/14-3-3ζ (H), and GPIbα/14-3-3ζ (I). Note the approximately equal contribution from each fluorochrome-conjugated secondary antibody at different fluorescence intensities in G (positive control), H, and I. This suggests that each computer pixel is detecting an equal amount of both antibodies present, which implies their respective epitopes are present in equal amounts (colocalized) in the 3-dimensional volume scanned by the microscope. This contrasts with F (1 of 2 negative controls), in which a diffuse pattern reflecting noncolocalization is seen. Three experiments with HEL cells and 2 with platelets were performed.