Figure 1.
MK budding is prominent in the bone marrow of mice. C57BL/6 mouse femoral bone marrow cryosections (10 and 40 μm) were immunostained with anti-CD41 (pink, for platelets and MKs) and anti-VWF (green, for granule content), co-stained with phalloidin (white, for F-actin), and then subjected to confocal and STED microscopy (Leica SP8; 93× glycerol objective; NA 1.3; z-step size, 80 nm). The images were deconvoluted using Huygens Software (ver. 20.04), processing was done using ImageJ (Ver. 1.53c), and surface renders were created using Imaris (ver. 9.8) using deconvolved fluorescent images. (A) Representative 2D confocal image (left panel) showing 2 MKs (MK1 and MK2) budding into a sinusoid. Their respective buds (Bud #1 and Bud #2), being released into the sinusoid (white) are indicated by arrow heads (▶). Buds were differentiated from transiting platelets by clear evidence of attachment to MKs based on the presence of CD41 joining the bud to the MK body. A released intrasinusoidal platelet is indicated by an arrow, which is not in direct proximity to a MK, differentiating it from a bud. Right panels depict a CD41+ STED optical section (top) and a 3D render created from the z-stack of the entire bud (bottom) of Bud #1 from MK1 (images were taken from one representative C57BL/6 bone marrow of n = 3, scale bar indicated). (B) Dot plot showing the size of buds and platelets in sinusoids, expressed as their maximal transverse diameter, from the evaluation of individual optical sections (50-60 buds and platelets from n = 3, C57BL/6 bone marrow), acquired using STED microscopy. (C) Representative confocal (left panel) and 3D STED render (right panel) images showing VWF+ buds (green; with granules) and VWF– blebs (without α-granules) on MKs. (D) Graph showing the percentage of VWF+ buds and VWF– blebs on MKs (64 budding MKs analyzed from n = 3, C57BL/6 bone marrows). (E) Graph showing the size of VWF+ granules within buds and platelets in the sinusoids. Data represent the mean granule size in 11 buds and 9 platelets. Statistical analysis (B, E) was performed using an unpaired t-test (GraphPad prism, v9.1.2).

MK budding is prominent in the bone marrow of mice. C57BL/6 mouse femoral bone marrow cryosections (10 and 40 μm) were immunostained with anti-CD41 (pink, for platelets and MKs) and anti-VWF (green, for granule content), co-stained with phalloidin (white, for F-actin), and then subjected to confocal and STED microscopy (Leica SP8; 93× glycerol objective; NA 1.3; z-step size, 80 nm). The images were deconvoluted using Huygens Software (ver. 20.04), processing was done using ImageJ (Ver. 1.53c), and surface renders were created using Imaris (ver. 9.8) using deconvolved fluorescent images. (A) Representative 2D confocal image (left panel) showing 2 MKs (MK1 and MK2) budding into a sinusoid. Their respective buds (Bud #1 and Bud #2), being released into the sinusoid (white) are indicated by arrow heads (▶). Buds were differentiated from transiting platelets by clear evidence of attachment to MKs based on the presence of CD41 joining the bud to the MK body. A released intrasinusoidal platelet is indicated by an arrow, which is not in direct proximity to a MK, differentiating it from a bud. Right panels depict a CD41+ STED optical section (top) and a 3D render created from the z-stack of the entire bud (bottom) of Bud #1 from MK1 (images were taken from one representative C57BL/6 bone marrow of n = 3, scale bar indicated). (B) Dot plot showing the size of buds and platelets in sinusoids, expressed as their maximal transverse diameter, from the evaluation of individual optical sections (50-60 buds and platelets from n = 3, C57BL/6 bone marrow), acquired using STED microscopy. (C) Representative confocal (left panel) and 3D STED render (right panel) images showing VWF+ buds (green; with granules) and VWF– blebs (without α-granules) on MKs. (D) Graph showing the percentage of VWF+ buds and VWF– blebs on MKs (64 budding MKs analyzed from n = 3, C57BL/6 bone marrows). (E) Graph showing the size of VWF+ granules within buds and platelets in the sinusoids. Data represent the mean granule size in 11 buds and 9 platelets. Statistical analysis (B, E) was performed using an unpaired t-test (GraphPad prism, v9.1.2).

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