Histological and Immunohistochemical Staining

Following euthanasia, all IVDs were obtained from each rat, fixed in 4% formaldehyde solution for 48 h, decalcified by 10% ethylenediaminetetraacetic acid (EDTA, Sigma) for 40 days, and finally embedded in paraffin. The paraffin-embedded IVD specimens were cut into 5-μm coronal sections with intact structures. Parts of these specimens were stained with safranin-O fast green and hematoxylin and eosin (H&E). The method established by Masuda was used to assess the histological morphology of the specimens (Table 2; Masuda et al., 2005).

Definition of histological scale.

TRAP staining was performed with a TRAP staining kit (#387A, Sigma-Aldrich, St. Louis, MO, United States). Dark purple particles were considered osteoclasts. A new method by Sawyer was used to quantify TRAP-positive cells (Sawyer et al., 2003). Immunohistochemical staining was used to observe specific expression of anti-Col-II (1:500, ab34712, Abcam), MMP13 (1:200, ab219620, Abcam), IL-1β (1:500, ab205924, Abcam), anti-p-P65 (1:200, ab16502, Abcam), and RANKL (1:500, ab239607, Abcam) in the rat IVD tissues. After being dewaxed, gradient hydrating and protease-modifying, sections were blocked with 5% horse serum and then incubated with the primary antibody overnight at 4°C. Sections were then washed with PBS, then successively incubated with secondary antibody and tertiary antibodies for 30 min, stained with 3,3′-diaminobenzidine tetrahydrochloride, and finally counterstained with hematoxylin. All images were taken using a high-resolution microscope. The positive staining cells and areas were analyzed using ImageJ software.