Hematoxylin–eosin stain and immunohistochemistry were performed as previously described [26 (link)]. Tissue sections were used for safranin O staining according to the standard protocol. Tissues were fixed in 4% paraformaldehyde for 48 h and incubated in 15% DEPC-EDTA (pH 7.8) for decalcification. Then, specimens were embedded in paraffin and sectioned at 7 μm. Immunofluorescence was performed as previously described [27 (link)]. Sections were blocked in PBS with 10% horse serum and 0.1% Triton for 1 h and then stained overnight with anti-PCNA antibody (SC-56). Donkey-anti-rabbit Alexa Fluor 488 (1:1000; Molecular Probes, A21206) was used as secondary antibodies. DAPI (Sigma, D8417) was used for counterstaining. Slides were mounted with anti-fluorescence mounting medium (Dako, S3023), and images were acquired with a Leica SP5 and SP8 confocal microscope. DIG labeled in situ hybridization (Roche) and immunohistochemical staining (Dako). OA Research Society International (OARSI) histopathological scores follow the literature that has been reported [28 (link)].
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