Sc 133192

Immunofluorescence staining of NP cells and the indicated IVD tissues was performed with anti-cortistatin (diluted 1:150, sc-393108, Santa Cruz Biotechnology), anti-TNFα (diluted 1:100, sc-133192, Santa Cruz Biotechnology), anti-Col 2 (diluted 1:100, sc-52658, Santa Cruz Biotechnology), anti-aggrecan (diluted 1:200, 13880-1-AP, Proteintech), anti-Annexin V (diluted 1:100, ab14196, Abcam), anti-NLRP3 (diluted 1:100, ab4207, Abcam), anti-IL-1β (diluted 1:200, ab9722, Abcam), anti-p65 (diluted 1:150, ab86299, Abcam) and anti-MMP13 (diluted 1:100, sc-515284) antibodies. The procedure was conducted as we described previously 35 (link), and images were taken with a fluorescence microscope (Olympus IX51, Japan). The immunofluorescence signal intensities were quantified with ImageJ software 41 (link).

Laser-irradiated mice colons were isolated, washed with ice-cold PBS, and fixed with 4% paraformaldehyde for 24 h. The fixed colons were dehydrated by consecutive incubations in 70%, 80%, 90%, 95%, and 100% ethanol for 2 h each, after which they were embedded in 100% paraffin overnight, and then solidified and sectioned at 4-μm thickness. The tissue sections were subsequently deparaffinized and rehydrated, and their endogenous peroxidase was blocked by 15 min of incubation in 3% hydrogen peroxide. Antigen retrieval was performed by boiling the sections in a solution of 10 mM sodium citrate and 0.05% Tween-20 (pH 6.0) for 15 min. Afterward, the sections were incubated with the primary antibodies overnight at 4 °C, rinsed with PBS containing 0.05% Tween-20, and incubated with the secondary antibodies (vector lab, Burlingame, CA, USA). Finally, the slides were counterstained with Mayer’s hematoxylin and mounted for microscopic analyses. The primary antibodies used were as follows: anti-Ki-67 (1:5000, Abcam, Cambridge, UK, ab15580) and anti- tumor necrosis factor (TNF)-α (1:100, Santa Cruz Biotechnology, Dallas, TX, USA, sc-133192).

The maxillary tissues were fixed with 10% formalin (Sigma Aldrich, USA), decalcified with 0.5 M ethylene-diamine tetra acetic acid (EDTA, pH 7.4), and paraffin-embedded. The specimens were sliced into 5 µm thick sections, and TRAP and hematoxylin-and-eosin (H&E) staining analyses were performed, as previously mentioned [2 (link)]. TRAP-positive cells were counted using Image J (Media Cybernetics, Inc. Rockville, MD, USA) [33 (link)].
To identify proinflammatory cytokine expressions, IHC analysis was performed with primary antibodies against IL-1β (AF-401-NA, R&D Systems, Minneapolis, MN, USA), IL-6 (ab6672, Abcam, UK), and TNF-a (sc-133192, Santa Cruz, CA, USA). For the secondary antibody reactions, VECTASTAIN^® Universal Quick Kit (PK-7800, Vector Laboratories, Burlingame, CA, USA) for IL-1β and Dako EnVision+ System Kit (K4009, Dako, Denmark) for IL-6 and TNF-a were used. The DAB Peroxidase Substrate Kit was used to visualize the cytokine expressions in the tissues [2 (link)]. The quantitative comparisons were conducted by calculating the percentage of positive staining on the same-sized area in the furcation of the maxillary second molar using Image J Fiji (Version 1.2).