Neutral gum

After paraffin embedding, a tissue section with a thickness of 4 μm was prepared in the Department of Pathophysiology, Guizhou Medical University. The tissue slices were heated at 65°C until the wax dissolved and immersed in xylene twice for 5 min each time. Tissue slices were then immersed in 100% alcohol twice, 95% alcohol twice, 80% alcohol once, 70% alcohol once, 50% alcohol once, and washed with running water twice, 2 min each time. The slices were immersed in hematoxylin for 10 min, then rinsed with tap water for 1 min, immersed in 1% hydrochloric acid alcohol for 5 s, and rinsed with lithium carbonate solution for 5 s to turn the slices blue. Next, the slices were immersed in 2% eosin alcohol for 3 min for coloration. 75% alcohol once, 80% alcohol once, 95% alcohol twice, 100% alcohol twice, and in dimethylbenzene twice, each time for 5 min. Finally, neutral gum (Beyotime Biotechnology, Jiangsu, China) was used as a sealant.

The maxilla samples were placed in 0.01 M phosphate buffered saline (PBS; pH 7.4) for 12 h and the solution was renewed twice over this time-period. The samples were enucleated and fixed in formaldehyde, then processed and embedded in paraffin (Leica Microsystems, Wetzlar, Germany) using routine procedures (13 (link)). At the maxillary first molar tooth, along the long axis of the proximal and distal length, tooth periodontal slices (6 μm) were cut.
The slices were dewaxed with xylene (Beyotime Biotechnology) and rehydrated with a descending graded series of alcohol. The slices were subsequently rinsed with tap water and hematoxylin-stained for 5 min. The slices were then rinsed, 1% diluted ammonia was added for 30 sec to retrieve the blue color, and the slices were again rinsed. The slices were stained with eosin for 5 min, rinsed, dehydrated with a graded series of alcohol, made transparent with xylene and finally mounted with neutral gum (Beyotime Biotechnology).

The mouse lumbar spines (L3–L6) were used for IHC staining. After deparaffinization and hydration, the slices were placed in the citrate buffer and heated to 95°C for 10 min; 3% H₂O₂ was then used to inactivate peroxidases. After blocking nonspecific antigens with 10% goat serum, we incubated the slices with primary antibody dilution solution at 4°C overnight. The primary antibodies used were as follows: PTH1R (1 : 100, ab75150, Abcam, USA), collagen II (1 : 300, ab34712, Abcam, USA), collagen X (1 : 100, ab58632, Abcam, USA), superoxide dismutase 1 (SOD1, 1 : 1000, ab51254, Abcam, USA), SOD2 (1 : 200, ab68155, Abcam, USA), proliferating cell nuclear antigen (PCNA, 1 : 300, ab92552, Abcam, USA), Ki67 (1 : 500, ab15580, Abcam, USA), caspase3 (1 : 1000, ab184787, Abcam, USA), caspase9 (1 : 300, ab202068, Abcam, USA), SHH (1 : 500, ab135240, Abcam, USA), and GLI1 (1 : 200, ab217326, Abcam, USA). The slices were then washed 3 times with phosphate buffer solution (PBS) and incubated with secondary antibody dilution (Goat Anti-Rabbit IgG, 1 : 500, 12-348, Sigma Aldrich, USA; Goat Anti-Mouse IgG, 1 : 500, 12-349, Sigma Aldrich, USA) at room temperature for 1 h. Diaminobenzidine was used to develop the colour. Finally, the cell nuclei were stained with haematoxylin (Beyotime, China), and the slices were mounted with neutral gum (Beyotime, China) after dehydration.