For histological analyses, the maxillae collected from experimental mice were fixed in 4% PFA and decalcified in 0.5 M EDTA for 3 weeks. Then, the maxillae were embedded in Tissue-Tek optimum cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA) and cut in a freezing microtome (Leica CM1900, Germany). Sections were stained with haematoxylin and eosin (H&E) and tartrate-resistant acid phosphatase (TRAP; Jiancheng Technology, Nanjing, China). TRAP-positive multinucleated (>3 nuclei) cells were considered osteoclasts [38 (link)]. The CEJ-ABC distance was measured to evaluate bone loss.
For IF staining, the maxillae from experimental mice were embedded in Tissue-Tek OCT and cut in a freezing microtome (Leica CM1900, Germany). Sections (5 μm thick) were placed on glass slides (CITOGLAS, Jiangsu, China), blocked with buffer containing 0.05% PBS-Tween and 0.5% FBS for 30 min, and sequentially incubated with the appropriate primary antibodies overnight at 4 °C and secondary antibodies for 60 min at room temperature. Then, sections were counterstained with DAPI for 3 min at room temperature. IF signals were visualized and recorded using a LSM780 laser scanning confocal microscope (Zeiss, Germany).
For IF staining, the maxillae from experimental mice were embedded in Tissue-Tek OCT and cut in a freezing microtome (Leica CM1900, Germany). Sections (5 μm thick) were placed on glass slides (CITOGLAS, Jiangsu, China), blocked with buffer containing 0.05% PBS-Tween and 0.5% FBS for 30 min, and sequentially incubated with the appropriate primary antibodies overnight at 4 °C and secondary antibodies for 60 min at room temperature. Then, sections were counterstained with DAPI for 3 min at room temperature. IF signals were visualized and recorded using a LSM780 laser scanning confocal microscope (Zeiss, Germany).