*4.7. Immunohistochemistry (IHC) for Type X Collagen (Col X), Indian Hedgehog (IHH), Matrix Metalloproteinases 13 (MMP13), Caspase 3, mTOR, Light Chain 3 (LC3), Beclin-1*

Tissue sections were deparaffinized, rehydrated and then blocked with 3% hydrogen peroxide. The samples were prepared for indirect immune detection by mouse- and rabbit-specific horseradish peroxidase/diaminobenzidine detection kit (ab64264, Abcam, Cambridge, MA, USA) following the manufacturer's instructions. The primary antibodies used in this study were rabbit polyclonal antibodies to Col X (ab58632, Abcam, Cambridge, MA, USA), IHH (ab52919, Abcam, Cambridge, MA, USA), MMP13 (ab39012, Abcam, Cambridge, MA, USA), activated caspase-3 (ab2302, Abcam,

Cambridge, MA, USA), phosphorylated-mTOR(Ser235/236)(4858s, Cell Signaling, Danvers, MA, USA), LC3 (14600-1-AP, Proteintech, Rosemont, IL, USA), and beclin 1 (11306-1-AP, Proteintech, Rosemont, IL, USA). The sections were counterstained with hematoxylin and the immunolocalized nuclei were stained in brown. For quantification, the sections of related target proteins staining were digitalized at 400 times magnification in a total of 2560 × 1920 pixel and 300 dpi digital images in JPG file format using TissueFAXS microscope (TissueGnostics GmbH, Vienna, Austria). The digital images were analyzed to quantify the total amount of related target proteins staining using HistoQuest (TissueGnostics, Los Angeles, CA, USA) analysis software. Total area of related target protein staining was detected after automatic color separation by HistoQuest. The staining intensity was measured as mean intensity of all pixels with an automatic background threshold range from 5 to 255. The results were given as percentage per mm<sup>2</sup> of total tissue area.
