Abt178

The paraffin sections were baked at 65 °C overnight. Slides were then deparaffinized and rehydrated. Dako endogenous blocking reagent (S2003, Dako, Carpinteria, CA, USA) was then used to quench endogenous peroxidase for 15 min. Non-specific binding sites were blocked with 1:10 normal horse/goat serum (S-2000, Vector Laboratories, Burlingame, CA, USA) for 30 min at room temperature. Primary antibodies: 1:400 dilution of MMP13 (ab39012, Abcam, Cambridge, UK), 1:1 000 dilution of ColX (ab49945, Abcam, Cambridge, UK); 1:400 dilution of Admts4 (ABT178, Millipore, Billerica, MA), and 1:500 dilution of Adamts5 (ab41037 Abcam, Cambridge, UK) were added, and the slides were incubated at 4 °C overnight. For IHC assays, the secondary biotinylated goat anti-mouse antibody (BA-9200, Vector Laboratories) at the dilution of 1:200 was added for 30 min on the second day, followed by incubation with 1:250 streptavidin (21130, Pierce, Rockford, IL, USA) for 30 min. Positive staining was detected by Romulin AEC Chromagen (Biocare Medical RAEC810L, Concord, CA, USA). For IF staining, an appropriate secondary antibody conjugated to a fluorescence probe was added, incubated at room temperature for 1 h, rinsed in PBS, and mounted in an anti-fading mounting media (Vector Laboratories, Burlingame, CA). Results were obtained using an Olympus BX43 upright microscope (Olympus Optical, Tokyo, Japan).

IHC analysis was performed using non-phospho (active) β-catenin (Ser45) (1:800 dilution; CAT#:19807, Cell signaling technology, USA), collagen II antibody (1:200 dilution; CAT#: ab34712, abcam, Britain).
IF analysis detected MMP13 antibody (1:300 dilution; CAT#: ab39012, abcam, Britain), Admts4 (1:400 dilution; CAT#:ABT178, Millipore, Billerica, MA), and Adamts5 antibody (1:500 dilution; CAT#: ab41037, abcam, Britain).