Histological Analysis of Joint Tissues

The joints collected for histology were fixed in 4% paraformaldehyde, decalcified in EDTA solution (0.25 mol/l, pH 7.4), and embedded in paraffin. Haematoxylin and eosin (H&E) and safranin-O/fast green staining were conducted with 6 μm-thick tissue sections. Immunohistochemistry was performed following a standard protocol. Briefly, tissue sections were regularly deparaffinized and rehydrated. The antigen was retrieved with citric acid buffer (pH 6.0) and microwave antigen retrieval for 15 minutes. After blocking with hydrogen peroxide and serum, the tissue sections were incubated with ANT3 antibodies (Abcepta) overnight at 4°C. HRP-labelled secondary antibody was incubated at 37°C for 60 minutes. The colour was developed by applying 3,3′-diaminobenzidine tetrahydrochloride (DAB) (DakoCytomation, USA), and the sections were counterstained with haematoxylin.^{24 (link)}