Mmp 13

IVD specimens were fixed, decalcified, dehydrated, paraffin-embedded, and processed into serial 4 μm coronal sections. After deparaffinization and hydration, hematoxylin and eosin (H&E), Safranin O/Fast Green (SO/FG) and Masson staining were performed. For immunohistochemistry (IHC), slices were placed in citric acid solution (50mM) and incubated in a water bath at 60°C for 16 h to extract antigen, then incubated in 30% hydrogen peroxide solution for 10 minutes and blocked with 10% goat serum at 37°C for 1 hour. The sections were incubated in antibodies against MMP-13, OCN, NCOA4, LGR5 (purchased from ABclonal), Collagen X, p53, p16^IKN4A, Ferritin, β-catenin (purchased from Abcam), and Col2a1 (purchased from Millipore) at 4°C overnight, followed by incubation in secondary antibody (purchased from Abcam) for 1 hour at room temperature, DAB chromogenesis and hematoxylin staining. For IF staining, the sections were incubated with a secondary antibody (purchased from Abcam) followed by staining with DAPI. In the quantitative analysis, the CEP height was calculated by using the mean value of the CEP height at 25%, 50%, and 75% of the coronal position of the IVD. The degree of IVDD was scored according to the modified scoring method in the previous literature.⁴⁷ (link) IHC and IF staining were performed to compare protein expression by calculating the positive cell ratio.