In some cases we injected retro-orbitally with 10 μg of Alexa Fluor 647 anti-mouse CD31 (BioLegend, no. 110724) and 10 μg of Alexa Fluor 647 anti-mouse CD144 (BioLegend, no. 138006) ten minutes before euthanasia to visualize the BM vasculature as described34 .
Whole-mount sternum stain has been described before38 . Sterna were processed immediately after euthanasia. After dissection we removed all connective tissue by gentle scraping with a blade. Fragments with bone marrow cavity were dissected and sectioned along the sagittal or coronal plane to expose the BM as described. Each half of the sternum was fixed in 4% PFA (Electron Microscopy Sciences, no. 15710) in DPBS for 3 hours. Each fragment was washed thrice with DPBS, followed by 1 hour blocking in DPBS containing 10% (v/v) goat serum (Sigma-Aldrich, no. G9023). We stained each sample with 100 µl staining buffer (2% goat serum in DPBS and the indicated antibodies). All steps were performed at 4ºC.
Whole-mount sternum stain has been described before38 . Sterna were processed immediately after euthanasia. After dissection we removed all connective tissue by gentle scraping with a blade. Fragments with bone marrow cavity were dissected and sectioned along the sagittal or coronal plane to expose the BM as described. Each half of the sternum was fixed in 4% PFA (Electron Microscopy Sciences, no. 15710) in DPBS for 3 hours. Each fragment was washed thrice with DPBS, followed by 1 hour blocking in DPBS containing 10% (v/v) goat serum (Sigma-Aldrich, no. G9023). We stained each sample with 100 µl staining buffer (2% goat serum in DPBS and the indicated antibodies). All steps were performed at 4ºC.