Ab1031

Free-floating brain slices were rinsed three times in PBS and digested with chondroitinase ABC (C3667, Sigma; for details, see immunohistochemistry) as enhancing step for the AB1031 aggrecan staining (AB1031, Millipore). The sections were then blocked with blocking solution (5% normal donkey serum, Jackson ImmunoResearch, UK and 0.25% Triton X-100 in PBS) for one hour at RT. The sections were incubated overnight at 4°C with a primary antibody (polyclonal rabbit anti-aggrecan 1∶1000, AB1031, Millipore; monoclonal mouse anti-chondroitin sulfate proteoglycan 1∶1000, protein core epitope, Cat-315/MAB1581, Millipore; monoclonal goat anti-parvalbumin, 1∶2500, PV235, Swant) diluted in blocking solution. Following three rinses with 2% normal donkey serum and 0.25% Triton X-100 in PBS, the sections were incubated with appropriate secondary antibodies (donkey anti-rabbit/mouse/goat antibody) conjugated with either Cy3 or DyLight 488 fluorescent dyes (Jackson ImmunoResearch, UK) at a dilution of 1∶400 in blocking solution for 90 minutes at RT. Fluorescent dyes were imaged using a confocal laser-scanning microscope (Zeiss LSM 510, Jena, Germany).

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.

Cells were scraped and lysed on days 3, 5 and 7 during differentiation in lysis buffer at pH 7.4, consisting of 10 mM Trisma-base, 300 mM NaCl, 2 mM EDTA and 0.5% Triton-X-100 with 1 mini protease inhibitor tablet. Samples were then sonicated 10 times at 50% amplitude for 20 s and centrifuged at 17000 g for 3 min at 4 °C. Protein concentration was determined using the bicinchoninic acid assay.
Proteins were denatured with 2× Laemmli buffer and boiled at 100 °C for 5 min. The samples were run on 10% sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel and blotted onto nitrocellulose membrane (ThermoFisher Scientific). The membranes were blocked with 10% non-fat dry milk in Tris-buffered saline with 0.2% Tween-20 (0.2% TBS-T) for 1 h. The following antibodies were used: anti-HAPLN1 (1:1000; Novus Biologicals NBP1–59150), rabbit polyclonal anti-ACAN (1:500; Merck AB1031), mouse monoclonal anti-TNR (clone #619 MAB1624, 1:500; R&D Systems), and mouse monoclonal anti-GAPDH (1:5000; Sigma). The blots were incubated in primary antibody overnight at 4 °C, and then incubated with horseradish peroxidase-coupled secondary antibodies (Invitrogen) at a dilution of 1:1000 for 1 h at room temperature (RT; 23 °C). They were then rinsed three times in 0.2% TBS-T, and the signals were visualized using the SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific).