For BM IVM, two sites were selected for imaging, namely, the calvarium (skull) and the tibia (leg), to assess potential differences in BM in long bones versus other bone structures. For analysis of the BM in the calvarium, following anesthesia, an incision was made in the skin overlaying the head, within a distance from the eyes to the base of the ears. This resulted in exposure of the skull, which was subsequently mechanically thinned. Measurements in the BM were made between the sagittal suture bifurcation and the intersection of sagittal and coronal sutures. Image postprocessing and analysis were performed using the Fiji software. For IVM of the tibia, mice were prepared as previously published (39). In summary, the tibia was exposed by removing the skin on top of the leg, followed by removal of the muscle with a scalpel. The bone was then mechanically thinned. An SP8-STED microscope, or an LSM5 microscope with the features previously described, was used for imaging, and a special mouse holder for the microscope stage was especially designed for this purpose using TopSolid, a CAD/CAM (computer-aided design/computer-aided manufacturing) system. This model was later three dimensionally (3D) printed for use at the SP8-STED microscope.

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