Rabbit anti il 1ra

The dewaxed slide was treated with Proteinase K (Sigma-Aldrich) for proteolytic digestion and 3% H₂O₂ for the elimination of endogenous peroxidase activity. After blocking with normal goat serum, the slides were incubated with primary antibody overnight at 4 °C, respectively. The primary antibodies used in this study include: rabbit anti-OCN (Abcam), rabbit anti-IL-8 (Abcam), rabbit anti-CCL5 (Abcam), rabbit anti-IL-1β (Abcam), rabbit anti-IL-1ra (Abcam), anti-CD68 (Abcam), rabbit anti-Phospho-Histone H3S10 (Abcam), and rabbit anti-TRPM7 (Abcam). The slides were then incubated with goat anti-rabbit secondary antibody and visualized using Diaminobenzidine (DAB) staining kit (Santa Cruz Biotechnology, Santa Cruz, USA) following the manufacturer’s instruction. Immunofluorescent staining was done using Alexa-Fluor 488 conjugated anti-rabbit IgG or Alexa-Fluor 647 conjugated anti-mouse IgG secondary antibodies (Thermo Fisher Scientific) and Hoechst 33324 (Thermo Fisher Scientific). Immunofluorescent images were captured using an LSM 780 confocal microscopy (Zeiss, Germany)

The protein from homogenized bone tissue from hamster were isolated using Trizol reagent (Thermo Fisher, USA) following the manufacturer’s instructions. The protein from cultured BMM was harvested using RIPA Lysis and Extraction Buffer (Thermo Fisher, USA) containing a 1% Phosphatase Inhibitor Cocktail (Thermo Fisher, USA). The concentration of protein was measured using the BCA Protein Assay Kit (Thermo Fisher, USA). A total of 20 µg of protein from each sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the polyvinylidene difluoride (PVDF) membrane (Merck Millipore, USA). Then, the membrane was blocked in 5% w/v bovine serum albumin (BSA, Sigma-Aldrich, USA) and incubated with blocking buffer-diluted primary antibodies overnight at 4 °C. The primary antibodies used include mouse anti-NFATc1 (Santa Cruz, USA), mouse anti-TRAP (Abcam), rabbit anti-Cathepsin K (Abcam), mouse anti-RANK (Abcam), rabbit anti-NF-κB p65 (CST), rabbit anti-IL-1β (Abcam), rabbit anti-IL-1RA (Abcam), rabbit anti-TNF-α (Abcam), rabbit anti-phospho-JNK (CST), rabbit anti-JNK (CST), rabbit anti-β-actin (Abcam). The protein bands were visualized by using HRP conjugated secondary antibodies and an enhanced chemiluminescence (ECL) substrate (Advansta, USA) and exposed under the Typhoon5 Biomolecular Imager 680 (GE Amersham, USA).

Immunohistochemical (IHC) staining was done on FFPE (formalin-fixed paraffin-embedded) tissue Sects. (4–5 μm) cut from TMAs using rabbit anti-IL-1RA (1:1000, Abcam, UK; ab124692). IHC was performed strictly in line with the standard streptavidin–biotin-peroxidase complex method, and the staining signals were visually quantified using a scoring system from 0 to 9. The scores were obtained by multiplying the intensity of signals with the percentage of positive cells (signal: 0 = no signal, 1 = weak signal, 2 = intermediate signal, and 3 = strong signal; percentage: 0 = 0%, 1 ≤ 25%, 2 = 25–50%, and 3 ≥ 50%). Low and high expressions were defined as scores of < 6 and ≥ 6, respectively. Two pathologists independently examined each specimen.