Reduced mobility of CD47 in the plasma membrane of Ox-RBCs. Untreated (viable RBCs) or Cu/ascorbate-treated (Ox-RBCs) RBCs were incubated sequentially with the anti-CD47 mAb miap430 and Cy3-conjugated goat anti–mouse IgG, followed by fixation in 3% paraformaldehyde and 0.01% glutaraldehyde. (A) RBCs adherent to poly-l-lysine–coated coverslips were analyzed by laser-scanning confocal microscopy. Z-stacks were obtained by scanning focal sections 0.5 μm apart throughout the cells. Scale bar represents 2 μm. Images are representative of 3 independent experiments. (B) The number of visible CD47 clusters was quantified in midsections of untreated RBCs or Ox-RBCs. Data are mean plus or minus SEM for 3 experiments with 50 RBCs analyzed in each experiment. **P < .01, using Student t test for paired comparisons.