Sc 47089

To analyse the macroscopic characteristics of articular cartilage, femoral condyles were obtained from normal and OA-induced rats (3, 5, and 10 days) and observed under a stereomicroscope (E2 4D Leica, Heidelberg, Germany). The cartilage samples were then cryopreserved with 10% PBS-sucrose for 24 h, cryosectioned (Leica CM 1100; Heerbrugg, Switzerland), mounted on gelatine-coated slides, and stored at −20°C for 2 days. The samples were hydrated in PBS, treated with 0.2% Tween 20 in PBS for 10 min, and preincubated with 0.2% IgG-free bovine serum albumin (Sigma Chemical, Germany) for 20 min at room temperature. The sections were incubated overnight at 4°C with anti-Lxn goat polyclonal antibody (1:70, sc-47089, Santa Cruz Biotechnology, Santa Cruz, CA, USA) followed by incubation with fluorescein isothiocyanate (FITC)-tagged anti-goat IgG (1:50 Zymed Laboratories, South San Francisco, CA) for 1 h at room temperature. The nuclei were counterstained with propidium iodide for 1 min (1:3000; Vector Laboratories, Burlingame, CA), and the samples were mounted with Vectashield. As a negative control, the primary antibody was omitted. Rat heart tissue was used as a positive control. The sections were analysed with confocal microscopy (TCP-SP2, Leica, Heidelberg, Germany). These experiments were performed in triplicate.

The cartilage samples of normal and OA-induced rats (3, 5, and 10 days) were frozen in liquid nitrogen, mechanically pulverized, suspended in lysis buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1 % SDS) with complete protease inhibitor cocktail, and homogenized in a polytron tissue grinder (POLYTRON® PT 2100). The samples were shaken for 2 h at 4°C and clarified by centrifugation at 10,000 × g at 4°C for 5 min. The protein concentration was measured with a Bradford assay. Subsequently, the obtained samples were dissolved in Laemmli buffer (which contained 1% SDS and β-mercaptoethanol) and boiled for 5 min. SDS-PAGE was performed using 5–20% gradient gels of 7 cm and 80 μg of protein per lane, after which the proteins were transferred onto nitrocellulose membranes by wet transfer at 350 mA for 2.5 h.
The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (TBS), pH 7.5, containing 0.1% Tween 20 (TBS-T) for 2 h at 37°C with gentle shaking and incubated overnight at 4°C with rabbit polyclonal anti-Lxn (1:1000; sc-47089, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Peroxidase-conjugated goat anti-rabbit was used as the secondary antibody (1:40,000; Jackson Immunoresearch Laboratories Inc. West Grove, PA, USA) for 2 h at room temperature. These experiments were performed in triplicate.