Histological Evaluation of Cartilage Markers

The specimens (n = 4) were embedded into paraffin and sectioned at 5 µm. The sections were subjected to standard Safranin-O staining as previously described [28] (link). Immunostaining of Collagen I, II, X, and osteocalcin was performed as previously described [28] (link). Primary antibodies used were: Biotinlyated collagen I and II antibodies (1∶400, Chondrex, WA), mouse collagen X antibody (1∶500, Sigma, MO), mouse osteocalcin antibody (1∶500, Abcam, MA). For collagen X and osteocalcin detection, biotinylated rabbit anti-mouse secondary antibody (1∶1000, Dako, CA) was used. The sections with the same procedure but omitting the primary antibody were used as a negative control.