Immunohistochemical (IHC) staining and immunofluorescence (IF) staining

Immunohistochemical (IHC) staining was performed to analyze the proteins expressed by chondrocyte-related genes. Tissue sections were processed for antigen retrieval by digestion with 0.05% trypsin at 37°C for 15 minutes. Tissue sections were incubated with primary antibodies to Sox9 (sc-166505) (Santa Cruz, USA), collagen type II alpha 1 (col2a1) (sc-517571) (Santa Cruz, USA), collagen type X (ColX) (ab58632) (Abcam, Cambridge, MA, USA), and matrix metalloproteinase 13 (MMP13) (sc-101564) (Santa Cruz, USA) overnight at 4°C. Then, sections were incubated with the secondary antibodies for 1 hour and developed in 3,3′-diaminobenzidine (DAB) chromogen (brown) (Invitrogen, USA). The sections were counterstained with hematoxylin, and observed by light microscopy (Leica, Germany).

For the immunofluorescence (IF) staining, the sections were stained with primary antibodies against runt-related transcription factor (RUNX2) (ab23981) (Abcam, Cambridge, MA, USA), Indian hedgehog (Ihh) (ab39634) (Abcam, Cambridge, MA, USA) and parathyroid hormone-related protein (PHrP) (sc-12722) (Santa Cruz, USA). Imaging was performed using a Leica fluorescence microscope. Quantification of the positively-stained cells was performed by microscopy.