Immunohistochemistry was performed on deparaffinized and rehydrated sections with specific primary antibodies: type II collagen antibody (anti-mouse: 1:400, ab34712, Abcam and anti-rabbit: 1:100, 08631711, MP biomedicals), aggrecan antibody (anti-mouse: 1:100, ab1031, Merck Millipore and anti-rabbit: 1:100, MA3-16888, Thermo Fisher Scientific) and NITEGE antibody (anti-mouse: 1:500, PA1-1746, Thermo Fisher Scientific and anti-rabbit: 1:50, MBS442004, My Biosource (San Diego-CA, USA) for the detection of aggrecan cleavage. All sections were counterstained using Mayer’s hematoxylin (RAL Diagnostic, Martillac, France). Tissue staining was viewed using Nanozoomer 2.0 Hamamatsu slide scanner (Hamamatsu Photonics, Hamamatsu, Japan). Immunostaining intensity for NITEGE was quantified by determining the relative intensity of the stained articular cartilage matrix as previously described33 (link). Briefly, images were converted into greyscale. The mean grey level values of 12 distinct regions of interest (50 × 50 pixels) from the femoral condyle and tibial plateau were determined. The value obtained was corrected by the mean of the gray levels of the extracellular matrix (10 × 10 pixels). Then, the corrected mean was subtracted from the blank (10 × 10 pixels). Finally, the value is expressed as fold increase over control condition. Measurements were performed using FIJI software.
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