Donatello

Five rats from each group were euthanized using general anesthesia on days 3, 7, and 14. After the maxillary left alveolar bones of the rats were removed, they were scanned with micro-CT (voltage: 85 kV, current: 200 μA, resolution ratio: 10 μm; SkyScan 1276; Bruker, Karlsruhe, Germany) for analysis of orthodontic tooth movement, tooth resorption, and trabecular structure. Next, the bones were fixed with 4% paraformaldehyde (Servicebio) for 24 h; decalcified with ethylene diamine tetraacetic acid (EDTA; Servicebio) for several weeks; dehydrated with 75%, 85%, 90%, 95%, and 100% ethanol (Servicebio), alcohol benzene (Servicebio), and xylene (Servicebio) in an automatic tissue processor (Donatello; DIAPATH, Shanghai, China); and embedded with molten paraffin (Servicebio) in an embedding machine (JB-P5; Junjie, Wuhan, China). After cooling and trimmed, these wax blocks were sectioned into 4-μm-thick slides along the sagittal direction with a pathology slicer (RM2016; Leica), which were then used for hematoxylin-eosin (HE) staining, TRAP staining, immunohistochemistry, and immunofluorescence.

Specimens of normal colon tissue were obtained from a patient affected by colorectal cancer who underwent curative surgical resection at the Ospedale di Circolo, ASST Sette Laghi (Varese, Italy). After collection (10 cm distant from the tumor), the tissue was divided in three pieces of similar size and fixed for 24 h, 48 h, or 72 h. All tissues were fixed in neutral-buffered formalin (formaldehyde 4% wt/vol and acetate buffer 0.05 M) and routinely processed in paraffin wax using the automated tissue processor Donatello (Diapath, Bergamo, Italy).