A2851

Femur tissue sections were blocked with 3% H₂O₂ for 1 h. After completely rinsing with 1 mol/L phosphate buffer (PBS), non-specific blocking serum was added and blocked at room temperature for 1 h. Then the primary antibody was added and incubated overnight at 4°C. The next day, after 3 times of washing with PBS, HRP Goat Anti-Rabbit IgG was added (A0208, Beyotime, China) and incubated at room temperature for 1 h. DAB was used for color development and the reaction was terminated by distilled water and then redyed with hematoxylin. Finally, the sections were observed, and graphs were captured with an inverted fluorescence microscope. Primary antibodies of Runx2 Rabbit pAb (A2851) and Anti-Sp7/Osterix antibody (Ab209484) were bought from Abclonal, China and Abcam, USA, respectively.

Fixed mouse maxillae were decalcified with 10% EDTA (pH 7.2) for 30 days and embedded in paraffin. Specimens were sliced at 5-μm thickness, followed by green fluorescent protein (GFP, ab1218, Abcam, UK), Runx2 (Runt-related transcription factor, A2851, ABclonal, China) and Lpl (lipoprotein lipase, A16252, ABclonal, China) immunohistochemistry, H&E staining (Beyotime, China) and TRAP staining (Solarbio, China).
In the HE staining, we selected a maximum width of the periodontal ligament from the cementum-enamel junction to the apex adjacent to the crown in the distal buccal root compression side. The width of the periodontal ligament was measured in this area.
In TRAP staining, a region of interest was selected for the number of osteoclasts from the cementum-enamel junction to the apex in the distal buccal root compression side of the first molar.
The region of one-third distance from the cementum-enamel junction to the apex adjacent to the crown was selected to determine the number of positive cells in the proximal buccal root of the first molar in GFP, GFP-Runx2 and GFP-Lpl double immunohistochemical staining for cell counting.

The cells were fixed with 4% paraformaldehyde for 15 min at RT. Then, cells were blocked with quickblock blocking buffer for immune staining (P0260, Beyotime, China) for 15 min at RT, followed by incubation with primary antibody against RUNX2 (1:200, A2851, ABclonal, China), or osterix (1:1000, ab209484, Abcam, UK) at 4 °C overnight and labeled with Alexa Fluor594-preabsorbed goat anti-rabbit IgG (ab150084, Abcam, 1:500, UK) for 2 h at RT. Next, the nucleus was stained with DAPI.