GP1bα-flnA interaction does not impair megakaryocyte budding in bone marrow. (A-F) Femoral bone marrow cryosections were immunostained with anti-CD41 (for platelets and MKs), anti-laminin (for sinusoids), and/or anti–P-selectin or anti-VWF (for granule stored content) antibodies and examined with confocal or STED fluorescence microscopy, as described under “Methods.” (A) Representative STED images showing MK budding into sinusoids, with the buds projected in 3D STED and bud size indicated by arrowheads (maximal transverse diameter). Scale bars of 10 μm (2D STED, left panel) and 3 μm (3D STED, right panel) and projected as a surface render using Imaris. (B) Graph showing percentage of hGPIbα^WT and hGPIbα^FW MKs undergoing budding in 10 μm thick BM cryosections (4 × 50 000 μm² fields per mouse were examined). (C) Number of buds per MK was determined for budding hGPIbα^WT and hGPIbα^FW MKs. Bud size (D) and sinusoidal platelet size (E) was determined using maximal transverse diameter on optical sections. A total of 35 to 40 MKs and platelets were assessed per mouse. Graphs represent mean ± SEM (n = 5-6 mice; ∗∗∗∗P < .0001). (F) The VWF containing buds in hGPIbα^WT and hGPIbα^FW mice were determined by immunostaining of bone marrow cryosections with anti-CD41 and anti-VWF Abs and confocal microscopy. The results are percentage of VWF⁺ buds over total buds examined (>20 CD41⁺ surface projections from each of n = 3 mice were examined). Ab, antibody; BM, bone marrow; SEM, standard error of the mean.