Microwave oven

Following the μCT imaging, samples were transferred to a microwave oven (Ted Pella, Redding, CA), in which a circulating 10 % ethylenediamine tetraacetic acid solution was held for decalcification. After a 2-week demineralization period, specimens were dehydrated through an ascending ethanol series then paraffin-embedded. 8μm-thick sagittal sections were cut and collected on Superfrost-plus slides for histology including Aniline blue, Masson’s Trichrome, Movat’s pentachrome, and Picro-sirius red staining, which followed published protocols [27 ].

Cells were seeded on 10 cm acrylic culture dishes (Corning) until 70% confluence, fixed in a cold solution containing4% formaldehyde (EMS), 2.5% glutaraldehyde (EMS), 100 mM cacocylate buffer (Sigma) for 1 min using a microwave oven (at 150 watts, Ted Pella), and additional 40 min at 4 °C. The samples were washed in the same buffer (3 × 15 min), post-fixed with 1% osmium tetroxide (EMS) and 1.2% potassium ferrocyanide in cacodylate buffer (100 mM) for 40 min, washed with buffer (3 × 15 min), stained with 1% uranyl acetate (EMS) for 40 min, and washed with Milliq water (3 × 15 min). The cells were scraped off the plates, transferred to 1.5 mL eppendorfs, and pelleted for 15 min at 15,000 rpm (Eppendorf Centrifuge model 5415R).The cells were dehydrated in an acetone series until 100% (3×) and embedded in an Epon resin. Ultrathin sections of 50 nm were obtained with a diamond knife (Diatome), collected on 300 mesh copper grids, and post-stained with UranyLess (EMS) for 5 min. Sections were observed in a Zeiss Libra 120 operated at 80 kV, and high-resolution images (4K × 4K) obtained with a CCD camera Gatan US4000.