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Publication protocol
Primary antibodies for cartilage and bone matrix breakdown products were received as a gift from Dr. John Mort and included anti-TEGE (aggrecanase mediated aggrecan cleavage neoepitope), anti-DIPEN (matrix metalloproteinase mediated aggrecan cleavage neoepitope) and anti-C1,2C (a collagen I and II cleavage neoepitope) and were utilized as previously described [28–31]. Additional antibodies were obtained from their various manufacturers: MMP13 (Proteintech), SOX9 (R&D Systems), phosphoERK1/2 (Cell Signalling). Sections were incubated for 15 minutes in 3% H2O2 in methanol to eliminate endogenous peroxidase activity, followed by blocking in 5% goat or donkey serum in PBS, and overnight incubation with primary antibody at 4°C or with no primary antibody for use as a control. Additional controls were performed with rabbit or goat normal IgG (Santa Cruz) under the same conditions (S3 Fig). Sections were incubated with the appropriate secondary antibody (goat anti-rabbit, or donkey anti-goat, Santa Cruz) conjugated to horseradish peroxidase (HRP) and developed using DAB+ chromogen (Dako Canada). Sections were counterstained with methyl green, dehydrated in solutions of increasing ethanol concentration ending in xylene, and cover slipped.
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