Rankl

Myostatin was provided by Phoenix pep (San Francisco, CA, United States). RANKL was provided by Raybiotech (Guangzhou, China). The Smad2 inhibitor LY209761 was supplied by Selleck (Houston, TX, United States).

The maxillae of each mouse collected at the endpoint were defleshed and soaked in toluidine blue solution. Stained maxillae were photographed using a dissection microscope (Nikon), and total alveolar bone loss was calculated by measuring the cemento-enamel junction (CEJ) to the alveolar bone crest decalcification (ABC) distances on the buccal side of each root [41 (link)]. Some maxillae were fixed in 10% formaldehyde and then 10% EDTA solution at 4 °C for 3 to 4 weeks. The decalcified maxillae samples were then embedded in paraffin and sectioned (7-μm thickness) for TRAP and hematoxylin and eosin (H&E) staining. For immunofluorescence staining, the decalcified maxillae samples were embedded in OCT compound (Fisher Scientific) and sectioned (6 μm thickness) using a cryostat. The expression of Sema4D was monitored using biotin-conjugated anti-Sema4D-mAb, followed by avidin conjugated to AlexaFluor 488. Gingival tissue homogenates were obtained from the gingival tissue of each mouse at the endpoint, and Sema4D, TNF-α, RANKL, OPG, IL-10 and IGF-1 production in the homogenate was analyzed using an ELISA kit purchased from the following sources: Sema4D: RayBiotech; TNF-α, RANKL, OPG, IL-10 and IGF-1: R&D Systems.