The cartilage samples of tibial plateau containing 2 cm thickness of subchondral bones were collected from the sacrificed rats for further immunohistochemistry (IHC) detection. The samples were fixed in 4% paraformaldehyde for 24 h, and then decalcified in 10% ethylene diamine tetraacetic acid (EDTA) (pH 7.3), dehydrated, and paraffin embedded. Slices of 5 µm were taken through the tissue samples at an interval of 50 µm. Safranin O staining was performed by using of Safranin O staining kit (Keygen, Nanjing, China) to evaluate the degree of chronic cartilage lesion according to the manufacturer’s instructions. IHC staining was executed to measure the expression of Col2a1/aggrecan, as previously described [75 (link)]. Briefly, slices were incubated with primary antibodies at 4 °C overnight and secondary antibodies at 37 °C for 30 min consecutively, and were subsequently incubated with streptavidin horseradish peroxidase, stained with 3,3-diaminobenzidine, then counterstained with hematoxylin, dehydrated in a graded ethanol series (absolute ethyl alcohol for 3 min, 95% ethanol for 3 min, and 85% ethanol for 3 min), and finally mounted.
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