Extended Data Fig. 5: CD200Hi capillaries preferentially localize at the metaphyseal endosteum.
From: Bone marrow niches orchestrate stem-cell hierarchy and immune tolerance
a, Confocal microscope images of the long BMs. Left: Z-projection of confocal image stacks of whole-mount BMs (sum of 61 slices; 3 µm steps; z = 180 µm in total). CD200 signals (magenta) at the metaphyeal endosteum are denser than those at the diaphyseal endoteum. CD200 signals are scarcely found in the central marrow region. BM endothelial vessels (green) are distributed throughout the BM cavity. Magenta: Q655-CD200. Green: Alexa 568-IB4. The scale bar indicates 200 µm. Right: confocal image demonstrating the vascular network at the metaphysis. CD200Hi capillary beds (magenta) surround transitional regions from BM arterioles (red) into EMCN+ type H vessels (cyan). Magenta: Q655-CD200. Cyan: Alexa 488-EMCN. Green: Alexa 568-IB4. Red: Cy3-Sca1. C: CD200Hi capillaries. A: Arterioles. H: Type H vessels. The scale bar indicates 20 µm. Refer to Supplementary Videos 1–3. b, Sca1 and IB4 signals in “fine” CD200Hi capillaries (<3–4 µm in diameter) confirmed by zooming high-resolution images (1024 × 1024 or 2048 × 2048 pixels) and/or by increasing signal amplitudes. Upper: confocal image of CD200Hi capillaries stained with Alexa 594-Sca1 antibodies (red) and Alexa 647-IB4 antibodies (green) but not with Alexa 488-EMCN antibodies (cyan). Red: Alexa 594-Sca1. Green: Alexa 647-IB4. Cyan: Alexa 488-EMCN. Magenta: Q655-CD200. The scale bar indicates 20 µm. Lower: maximum intensity z-projection of confocal image stacks of whole-mount BMs achieved by selecting the highest signal value of each pixel along the z-axis (9 slices; 1 µm steps; z = 9 µm in total). Confocal image of CD200Hi capillaries stained with Alexa 568-Sca1 antibodies (red) and Alexa 647-IB4 antibodies (green) but not with Alexa 488-EMCN antibodies (cyan). Red: Alexa 568-Sca1. Green: Alexa 647-IB4. Cyan: Alexa 488-EMCN. Magenta: Q655-CD200. The scale bar indicates 50 µm. CD200HiIB4+Sca1+capillaries (C) surround Type H vessels (H) and arterioles (A). Refer to Supplementary Fig. 2a–h. c, Confocal image of Q655-CD200Hi capillaries co-stained with anti-CD31 antibodies. Green: CD31-APC. Magenta: Q655-CD200. The scale bar indicates 20 µm. d, Confocal image of Q655-CD200Hi capillaries in tamoxifen-treated VECadcreERT2 × Rosa26TdTomato mice. Tamoxifen treatments (intraperitoneal; 1 mg) were performed on Days 0–4, and BMs were isolated on Day 7. Amongst TdTomato+ BM endothelial cells (green), Q655-CD200 signals (magenta) identify fine capillaries (<2–3 µm in diameter) on the metaphyeal endosteum. Green: VECadcreERT2 × Rosa26TdTomato. Magenta: Q655-CD200. The scale bar indicates 20 µm.
