Femurs were dissected and cleaned from muscle, and placed immediately in 4% PFA at 4°C for 30 min. Subsequently, femurs were washed three times in 1X PBS before incubating with 15% sucrose for 2 h, followed by 30% sucrose for another 2 hours. All incubations were performed at 4 °C. Femurs were placed in OCT and frozen at -80 °C until sectioning.
For sectioning, a cryotome (ThermoFisher) was used to generate 12 μm sections at -20 to -22 °C, which were then transferred to slides using the CryoJane Tape-Transfer System (Leica Biosystems). Slides were post-fixed with 4% PFA for 1 min to enhance the adherence of sections to the slide, then washed for 2 min by dipping in PBS. Sections were stained with antibodies in blocking buffer (PBS containing 10% goat serum and 0.2% Triton X100, seetable S4 for antibodies used) at 4°C overnight, then washed by dipping into PBS for 1 min. Secondary antibody staining was done in blocking buffer at room temperature for 2 h. Sections were imaged using an LSM710 microscope (Zeiss) equipped with a polychromatic META detector (Lasers: 458, 488, 514, 561, 594, and 633nm), and an Olympus FV3000 Confocal laser scanning microscope with 4 GaAsP spectral detectors, FRAP and FRET (Lasers: 405, 445, 488, 514, 561, 594 and 640 nm). Imaris software (v8.41) was used for data analysis and representation.
For sectioning, a cryotome (ThermoFisher) was used to generate 12 μm sections at -20 to -22 °C, which were then transferred to slides using the CryoJane Tape-Transfer System (Leica Biosystems). Slides were post-fixed with 4% PFA for 1 min to enhance the adherence of sections to the slide, then washed for 2 min by dipping in PBS. Sections were stained with antibodies in blocking buffer (PBS containing 10% goat serum and 0.2% Triton X100, see