Ab233080

The RIPA buffer (Solarbio Life Sciences, China) was used to extract the total protein of samples from IVD tissues, according to the protocol. The BCA Protein Assay Kit (Thermo Fisher Scientific) was used to measure protein contant. Approximately 25 μg of total protein was boiled, separated using 12% SDS-PAGE, and blotted onto PVDF membranes. Then the membranes were sealed with 5% skimmed milk for 2 h, incubated with primary antibodies against IGF-1 (1:1000, ab223567, Abcam, Cambridge, MA, USA), TGF-β (1:500, ab92486, Abcam), SDF-1 (1:1000, ab18919, Abcam), CCL-5 (1:1000, ab189841, Abcam), SOX9 (1:1000, ab26414, Abcam), Aggrecan (1:1000, ab3778, Abcam), Collagen I (1:1000, ab233080, Abcam), Collagen II (1:1000, ab188570, Abcam) and GAPDH (1:5000, ab22555, Abcam), followed by incubation with horseradish peroxidase-conjugated secondary antibody for 2 h at room temperature. Blots were visualized using a chemiluminescence reagent (Millipore, USA). GAPDH was the internal reference. The relative expressions of proteins were normalized to GAPDH using Image-Pro software. The antibody information used in this study were as follows.Western blot experimental results of the original film in the supplementary file 1.

The collected specimens of the mandible (1.0 cm long, 1.0 cm wide and 1.0 cm thick bone blocks) were fixed in neutral formalin for one week, decalcified in chelaton for one month, dehydrated through a series of 70–100% ethanol solutions and embedded in paraffin. The specimens were then serially sectioned at 7 μm thickness in the sagittal plane, mounted on slides and prepared for histological (haematoxylin and eosin staining) and immunohistochemical analysis. The physiological bone sections from the left side of the mandible were used as reference control samples.
An immunohistochemical reaction was performed to demonstrate the presence of collagen I using a primary rabbit polyclonal anti-collagen I antibody (Abcam, ab233080) and a secondary DB DET SYS kit, the DB detection kit—rabbit/mouse dual system (Biotech). DAB (3,3′-diaminobenzidine) (DAKO) was used to visualize the reaction. Finally, the cell nuclei were stained with acidic Mayer’s haematoxylin.