Ab235957

Heterogeneous SF-SCs were isolated from the femurs of a 6–8-week-old sheep fetus (obtained at the local abattoir) by flushing with L-15 medium supplemented with Anti-Anti (Thermo Fisher Scientific). Cells were then expanded in vitro and verified for multipotency according to differentiation methods for chondrogenesis^{61 (link)}, osteogenesis^{62 (link)}, and myogenesis^{63 (link)}. Differentiated and undifferentiated SF-SCs were fixed in 4% buffered formaldehyde and stained with a wide range of primary antibodies (1 h at room temperature) from Abcam (Amsterdam, Netherlands), including α-SMA (ab32575; 1:500), vimentin (ab8798; 1:100), CD166 (ab235957; 1:200), Ki67 (ab15580; 1:300), estrogen receptor-α (ER-α; ab66102; 1:100), ER-β (ab187291; 1:100), progesterone receptor (PR; ab2765; 1:100), cytokeratin (ab9377; 1:1000), MyoD1 (ab16148, 1:100), RANK (ab13918, 1:100), and DMP1 (ab103203, 1:100). Each primary antibody was conjugated with either CY3 or Alexa Fluor 488 secondary antibodies (Thermo Fisher; 1:300) and DNA was labeled with DAPI. Chondrogenesis was evaluated after alcian blue staining using standard methods.

Immunofluorescence staining was used to analyze the distribution of MSCs in the intervertebral disc. Antigen retrieval was performed by soaking deparaffinized sections in a solution containing sodium citrate for 20 min at a 90 °C water bath. The slices were blocked with 5% BSA solution, incubated with 1:10 diluted CD90 and CD166 primary antibodies (ab225 and ab235957, Abcam) for 12 h at 4 °C, followed by incubation with 1:200 diluted Alexa-fluor 488 and Alexa-fluor 647-labeled secondary antibodies (A32723 and A32733, ThermoFisher) for 1 h at room temperature. DAPI staining was used to display the cell nucleus. Immunofluorescence staining images were observed by confocal microscope (SP5, Leica), and analyzed by LAS X software (version 3.7.4.23463, Leica). For each immune stained section, the cells in the bone marrow and growth plate of vertebra were used as positive controls for CD90 & CD166 co-positive cells, while osteocytes in EP were used as negative controls. The number of CD90 & CD166 co-positive cells in NP cavity of each sample was counted based on the 20× Immunofluorescence images from three parts of the nucleus cavity. At least four parallel animal samples were used for each data of the experimental groups.