Phase-contrast and immunofluorescence analysis of megakaryocytes cultured in vitro from CD34⁺ peripheral blood samples from patient 1. (A) Phase-contrast microscopy shows that megakaryocytes from a control donor at day 12 of culture have typical long and discrete proplatelets (arrows). (B) In contrast, megakaryocytes from patient 1 give rise to proplatelets that are intertwined (arrowheads). (C-E) Fixed and permeabilized cells from the patient were subjected to immunofluorescence labeling for VWF (green). Results show that at day 12 for the patient, VWF has both intracellular and surface staining (white arrows). Nuclei are shown in blue by DAPI. (F-K) Confocal microscopy shows the distribution of VWF and P-selectin in cultured megakaryocytes from patient 1. Cells taken at day 12 were fixed and permeabilized prior to being labeled for VWF (green fluorescence) (F,I) and P-selectin (red fluorescence) (G,J). When the colors were merged (H,K), much of the VWF has a different localization to the α-granule marker, P-selectin, with the labeling for VWF clearly extending to the surface of the cell. Images were acquired using a 63 ×/1.4 NA oil objective.