Nb600 504

Cell aggregates were fixed in 4% paraformaldehyde at 4°C and embedded in paraffin. Next, they were cut into 6 μm thick sections, dehydrated with alcohol, and stained with safranin O and toluidine blue (TB). The slides were photographed by an optical microscope (Olympus BX51; Olympus, Tokyo, Japan).
Immunohistochemistry staining was used to characterize the density of the chondrogenic proteins of COL II and ACAN. After 21 days of culture, the aggregates were fixed in 4% paraformaldehyde at 4°C and 6 μm thick sections were prepared. After dehydration with alcohol, antigen retrieval was performed with 0.05% trypsin for 30 min at 37°C. The sections were blocked with 1.5% goat serum in PBS and treated overnight at 4°C with rabbit anticollagen type II (250484, Abbiotec, USA) and ACAN (NB600-504, Novus Biologicals, USA) primary antibodies. Next, they were incubated for one hour with anti-mouse (A2554, Sigma-Aldrich, Germany) and anti-rabbit (ab97051, Abcam, UK) secondary antibodies. Images were acquired by an optical microscope (Olympus BX51; Olympus, Tokyo, Japan), and the density of the staining was assessed by Image-Pro Plus 6 software.

Expression of the hedgehog signaling molecules Patched (Ptc), Smoothened (Smo), and Gli1 was determined by western blotting. Expression of the cartilage-related proteins collagen II and ACAN was determined by western blotting following induction on days 10 and 21. Three samples from each group were randomly selected to extract cellular proteins. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control protein. The primary antibodies for Ptc (1:500, orb19265) and Smo (1:1600, orb19363) were purchased from Biorbyt (Cambridge, UK). Gli1 (1:1000, ARP32368_T100) was purchased from Aviva Systems Biology (San Diego, CA, USA). Primary antibodies against Runx2 (1:500, ab23981) and collagen X (1:500, ab58632) were purchased from Abcam (Cambridge, UK). Primary antibodies against collagen II (1:200, NB600–844), ACAN (1:100, NB600–504), and GAPDH (1:2000, NB300–328) were purchased from Novus Biologicals (Littleton, CO, USA). HRP-conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG, and HRP-conjugated anti-goat IgG antibodies were used as secondary antibodies at a 1:2000 dilution and were obtained from Thermo Fisher Scientific (Waltham, MA, USA).