Iv100 microscope

Sterna were harvested and processed as described previously^{6 (link)}, sectioned longitudinally and stained with rabbit anti-mouse tyrosine hydroxylase antibody (Millipore). Samples were incubated with secondary antibody and imaged using an Olympus IV100 microscope.

LKS cells were isolated from CD45.1STEM donor mice as described in the previous section. Freshly isolated LSK cells were labeled with a Vibrant™ DiD cell-labeling solution (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate) (Thermo Fischer Scientific, V22887) for 15 min at 37degree in the dark according to manufacturer instructions. 3 × 10⁴ LKS cells and 0.2 × 10⁶ supportive bone marrow cells were transplanted to lethally irradiated (9.5Gy) recipient mice via retro-orbital injection. 48hours after LKS cells injection, mice were anesthetized by isoflurane inhalation with 2% O_2. To visualize the bone structure, mice were previously administered intravenously with Osteosense® 750EX (4nmol/mouse, Perkin Elmer). To visualize the vasculature, mice were injected intravenously with anti-mouse CD31-AF647 (5μg/mouse), anti-mouse VE-Cadherin-AF647 (5μg/mouse) and anti-mouse Sca1-APC (2μg/mouse) antibodies (Key resource table) in 100ul of sterile PBS. The calvarium of the mice was imaged using an Olympus IV100 microscope (4X and 20X objective, Z-Stack of 2um). All images were analyzed and stitched using ImageJ software and Did+ cells were enumerated.