Anti col10a1

After washing paraffin sections with PBS, they were incubated in hydrogen peroxidase for 10 min to minimize nonspecific background staining. To detect chondrogenic markers, the sections were incubated with anti-COL2A1, anti-AGGRECAN, and anti-COL10A1 (1:100, Santa Cruz Biotechnology) antibodies at 4 °C for at least 12 h. The attachment of secondary antibodies and fluorescent protein-conjugated secondary antibodies was performed following previously described methods^{59 (link)}. Nuclei were stained with 4ʹ,6-diamidino-2-phenylindole (Sigma). An inverted fluorescence microscope (IX-71; Olympus, Tokyo, Japan) was used to acquire images and expression levels were quantified using ImageJ.

Both proliferative and hypertrophic MCT cells and ATDC5 cells under designated differentiation days were harvested, homogenized, and lysed in RIPA buffer containing proteinase inhibitor. After centrifugation, supernatant containing protein extracts were calculated and equal amount of proteins (100 μg) were used to run on SDS-PAGE gel (10 %), and then transferred onto PVDF membranes. After blocked with 5 % nonfat milk in TBS/T for 1 h, membranes were incubated with the primary antibodies anti-Cox-2 (D223097, Biotechnology, Shanghai, China) and anti-Col10a1 (sc-323750, Santa Cruz, CA, USA) at 4°C overnight. After washing, the membranes were then incubated with horseradish peroxidase–conjugated secondary antibody (goat anti-rabbit IgG antibody, D110058, Biotechnology, Shanghai, China) for 1 hour and subjected to detection using an enhanced chemiluminescence system (Minichemi, China). Anti-β-actin antibody was used in parallel as the loading control and experiments were done for three times to ensure conformance for the western assay.