Double-labeling confocal microscopy of the cellular localization of GPIIb, GPIbα, GPV, and GPIX at different stages of MK maturation.

Representative cells are shown for days 0, 4, 7, 10, and 14 of culture. The cells were permeabilized and labeled with anti-GPIIb revealed by a Cy3-GAM, anti-GPIbα–Alexa 488 or –Cy3, anti-GPV revealed by an Alexa 488–GAM and anti-GPIX–Cy2. Colored lettering matches the dye used for the corresponding fluorophore: red for Cy3 and green for Cy2 or Alexa 488. Confocal immunofluorescence images were recorded with a minimum pinhole size in the plane of the nuclei, using excitation and emission filtering as described in “Materials and methods.” Images in the rhodamine (Cy3) and fluorescein (Cy2 or Alexa 488) channels were recorded simultaneously in the same focal plane by a double exposure procedure.