Sc 30073

Tissue processing, section staining and counting of immune-positive cells were performed as reported previously21 (link)22 (link)23 (link)24 (link)25 (link). The primary antibodies were goat polyclonal α2A-adrenoreceptor (1:75; sc1478, Santa Cruz Biotechnology, Inc., USA), rabbit polyclonal β2-adrenoreceptor (1:100, ab137494; Abcam, Cambridge, United Kingdom), rabbit polyclonal aggrecan (1:200, ab36861, Abcam), goat polyclonal MMP-3 (1:50, sc6839, Santa Cruz), MMP-13 (1:50, sc30073, Santa Cruz), RANKL (1:50, sc7628, Santa Cruz). Six squares were applied at the quartering points of the central (each 0.15 mm × 0.15 mm) and posterior (each 0.2 mm × 0.2 mm) third of the condylar cartilage. Within the selected frames, the number of immune-positive cells and the percentage area of aggrecan-positive staining were determined. In the isotype control slides, isotype antibodies were substituted for the primary antibodies.

Conditioned media were concentrated by TCA precipitation and solubilized in Laemmli sample buffer. An equal amount of proteins were separated under reducing conditions on a 10% or 12% polyacrylamide gel. Resolved proteins were transferred onto a Hybond-C nitrocellulose membrane (GE Healthcare, München, Germany) in a semidry transfer system. The transfer efficiency was assessed by staining the membrane with Ponceau Red (Sigma-Aldrich, Diesenhofen, Germany). Membranes were blocked for 1 h with 5% milk powder and incubated overnight at 4 °C with the primary antibody. Primary antibodies used were anti-ADAM9, anti-decorin, (AF949 and MAB143, respectively, all from R&D Systems, Wiesbaden, Germany), anti-collagen type I (1:500; 20315035505 from Quartett, Berlin, Germany), anti-fibronectin and anti-actin (F3648 and A2668, both 1:1000; from Sigma Aldrich, Diesenhofen, Germany), and anti-MMP-13 (sc-30073, from Santa Cruz, Heidelberg, Germany; 1:1000), anti-MMP-14 (1:500) [18 (link)]. After washing, membranes were incubated with HRP-labeled secondary antibody (from Dako, Hamburg, Germany) for 1 h at room temperature. Bound secondary antibodies were detected by ECL^® system (Thermo Scientific, Bonn, Germany) and the membranes exposed to X-ray films (Thermo Fisher, Karlsruhe, Germany).