Anti aggrecan

MSC-laden constructs were harvested on d1 and d21 and fixed in 3.7% PBS-buffered formaldehyde over night at 4 °C. TissueTek^® O.C.T. (Sakura Finetek, Torrance, LA, USA) was used for embedding and 8 µm cryo-sections were performed at a cryostat (CM 3050S, Leica, Wetzlar, Germany). GAG deposition was visualized with safranin O (counterstain: Weigert’s hematoxylin and fast green), and collagen deposition with picrosirius red (counterstain: Weigert’s hematoxylin) [60 (link),61 (link)]. Immunohistochemical stainings were performed as previously described [38 (link)]. Used antibodies were: anti-aggrecan, 1:300, 969D4D11, Thermo Scientific, Waltham, MA, USA; anti-collagen I, 1:200, ab34710, Abcam, Cambridge, UK; anti-collagen II, 1:1000, II-4C11, Abnova, Taipei, Taiwan; and goat-anti-mouse Alexa488, 1:400, 111-545-003, Jackson ImmunoResearch, Cambridge, UK); all in 1% BSA in PBS. Cells were counterstained with DAPI during mounting (Dako, Hamburg, Germany).

At 6 and 12 weeks after transplantation, rats were sacrificed and the femoral trochlea were harvested. Frozen sections were stained by IHC using anti-Collagen II (ab34712, Abcam, UK), anti-Aggrecan (MA3-16888, Thermo Fisher, USA) and anti-Collagen I (380760, ZenBio, CHN) at 4 °C overnight, and horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology, USA) at 37 °C for 1 h. Sections were also stained for histological evaluation using safranin O-fast green staining. The residual immature area (RIA) and residual gel area (RGA) at 6 weeks were calculated by ImageJ V1.8.0 (National Institutes of Health, Bethesda, MD, USA). The cartilage regeneration at 3 months was evaluated by the Pineda score [13 (link)].

Samples were fixed overnight in 10% formalin. Samples were washed with PBS three times and then stained with alcian blue staining (Sigma-Aldrich, USA) to detect sulfated GAG. For IHC analysis, the primary antibody anti-aggrecan (Thermo Scientific, USA) was placed on the sections overnight at 4 °C. The sections were washed in PBS and incubated with a secondarybiotinylated antibody (Thermo Scientific, USA) for 30 min at room temperature. Immunostaining was detected by streptavidin-horseradish peroxidase (Thermo Scientific, USA), followed by colorimetric detection using DAB (Thermo Scientific, USA).