Immunohistochemical Analysis of Cartilage Matrix

Immunohistochemistry was performed on deparaffinized and rehydrated sections with primary antibodies directed against type II collagen (mouse antibody #CP18L, Calbiochem, France), antibodies directed against aggrecan (mouse antibody #MA3-16888, ThermoFisher, USA), and NITEGE (mouse antibody #MBS442004, My Biosource, USA) for the detection of aggrecan cleavage. First of all, antigen retrieval was carried out by incubation in either proteinase K (30 ​min 37 ​°C, 20 ​μg/mL #P6556, Sigma Aldrich, USA) or citrate buffer pH6 (10 ​min 100 ​°C or 3 ​h 70 ​°C) followed by incubation in hyaluronidase (15 ​min 37 ​°C, 1 ​mg/mL, #H3506, Sigma Aldrich, USA), for type II collagen and NITEGE immunostaining, respectively. For aggrecan immunostaining, antigen retrieved was carried out by chondroitinase (30 ​min at room temperature, 0.25U/mL, #C2905, Sigma Aldrich, USA) after reduction by dithiothreitol (2 ​h 37 ​°C, 10 ​mM, #D9760, Sigma Aldrich, USA) and alkylation by iodoacetamide (1 ​h 37 ​°C, 40 ​mM, #I1149, Sigma Aldrich, USA). Sections were then incubated with 3% (v/v) H₂O₂ (Sigma Aldrich, USA) to inactivate internal peroxidases. After blocking with 2.5% (v/v) horse serum (ref 30 ​022 #MP-7402 Vector Labs Burlingame, USA) for 30 ​min, sections were incubated overnight at 4 ​°C with the primary antibody solution (0.5 ​μg/mL for type II collagen, 10 ​μg/mL for anti-aggrecan, and 2 ​μg/mL for NITEGE in 0.1% (w/v) BSA). The sections were then incubated with peroxidase horse anti-mouse secondary antibodies (ref 30 ​028 #MP-7402, undiluted, Vector Labs) for 30 ​min at room temperature. The sections were developed with diaminobenzidine (DAB, #SK-4105, Vector Labs) for 3 ​min and counterstained using Mayer's hematoxylin (RAL Diagnostic, Martillac, France). Tissue sections were observed using Nanozoomer 2.0 Hamamatsu slide scanner (Hamamatsu Photonics, Hamamatsu, Japan) and imaged with NDP.view2 software® (Hamamatsu Photonics). Type II collagen, aggrecan, and NITEGE immunostaining were semi-quantified using QuPath® software [43 (link)] by measuring the diaminobenzidine (DAB) mean optical density (OD) in the articular cartilage matrix (20 measurements) and using the DAB mean OD in subchondral bone staining, as a blank (3 measurements). Then, the OD values were normalized to the mean intensity of cartilage in CL-sham joints. The results were expressed as an intensity ratio.