P 8811

Collagen 2 immunohistochemistry was performed as previously described58 (link) using mouse monoclonal anti-collagen 2 antibody (II-II6B3; DSHB Iowa) with Dako EnVision detection kit. In brief, slides were treated with 0.1% pronase (P-8811; Sigma) in PBS for 20 minutes at room temperature, followed by 2.5% hyaluronidase (H-3506, Sigma) in PBS for 30 minutes at 37 °C. Endogenous peroxidase activity was quenched with 3% H₂O₂ for 10 minutes at room temperature. Nonspecific binding sites were blocked with 10% goat serum in PBS for 60 minutes at room temperature. Sections were then incubated with primary antibody against collagen type 2 diluted 1:50 in PBS overnight at 4 °C. The slides were then washed in PBS, and incubated with the secondary antibody (Dako Real Envision/HRP, K5007) for 30 min at room temperature. DAB (Dako Real DAB+Chromogen, K5007) was applied for about 2 min and then removed by rinsing with distilled water. Slides were mounted with Biomount Aqua (Biognost, Croatia) and covered. Exclusion of primary antibody resulted in no staining.

The experimental protocol and retrieval process of human tissues were reviewed and approved by the Research Ethics Committee of Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation (IRB106-48-A). Written informed consent was obtained from all patients. Articular cartilage samples were harvested from 12 patients with advanced knee OA (five women and five men; age range, 67–84 years; average age, 75.8 ± 6.2 years). An additional two women and one man (age range, 63–80 years; average age, 70.7 ± 8.6 years) were enrolled for a Sirt1 study. The collected cartilaginous tissues were treated with 0.1% protease (P8811, Sigma-Aldrich, Saint Louis, MO, USA) for 30 min and then digested using 0.2% type II collagenase (9001-12-1, Gibco, USA) overnight for chondrocyte isolation [17 (link)]. The released cells were maintained in Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (10565018, Gibco) supplemented with 10% fetal bovine serum (SH30396.03, Hyclone, USA) and 1% antibiotic (15140-122, Gibco) in an incubator at 37 °C in a humidified atmosphere with 5% CO₂. Cells obtained from each donor were cultured at passages 2–6 and studied independently.