All animal work was conducted strictly following institutionally approved animal protocols. Closed transverse diaphysis fractures of the right femur were generated at the mid-femur using a drop-weight blunt guillotine device, in 2-month-old mice as previously described [24 (link)], but in immune-deficient NOD/SCID IL2Rγ–/– (NSG) mice. One hour after causing fracture, tdTomato-expressing human MSCs (treated with or without Kifunensine) were injected at 500,000 cells (resuspended in 20 μL PBS) per mouse, near the fracture site. This study was performed with four mice per condition. Two additional mice were injected with PBS only, to serve as negative control (to determine background fluorescence). After three days, mice were humanely euthanized, and samples fixed with formalin for 24 h, decalcified using 0.5 M EDTA (pH 8.0, USB Corporation, Cleveland, OH, USA) for an additional 24 h, and embedded in optimum cutting temperature (OCT) for cryosectioning. Cells were directly visualized based on tdTomato expression and DAPI staining for total nuclei. Sections were imaged using a BioRevo Keyence BZ-9000 fluorescence microscope (Keyence, Itasca, IL, USA) at ×10 for stitched images (low magnification) and at ×20 for high magnification images.
Free full text: Click here
