Gb11113

Knee joint samples were fixed in 10% paraformaldehyde for 24 h and then were treated with Ethylene Diamine Tetraacetic Acid (EDTA) until decalcification was complete. The decalcified specimens were dehydrated, embedded in paraffin, and sectioned at a 4 μm thickness. Next, the slices were processed for hematoxylin and eosin (H&E) and Safranin O/Fast Green staining. For immunohistochemical (IHC) staining, the sections were separately incubated with type 2 collagen (COL II) (1:300, GB11027, Servicebio), MMP13 (1:200, GB11247, Servicebio), ADAMTS5 (1:100, DF13268, Affbiotech), CD14 (1:1000, GB11254, Servicebio), CD206 (1:500, GB13438, Servicebio), IL-10 (1:500, GB11108, Servicebio) and IL-1β (1:800, GB11113, Servicebio), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:200, GB23303, Servicebio). Finally, the staining color was developed using the DAB Detection Kit (Servicebio).
The Osteoarthritis Research Society International (OARSI) score was applied to evaluate cartilage degradation (Supplementary Table 1).22 (link) H&E and Safranin O/Fast Green staining were observed in a low-magnification field. The IHC staining was measured using the %Area parameter or the percentage of pixels in the image or selection highlighted in red using Image→Adjust→Threshold. Each parameter was graded and independently measured by two observers.

The Elivision Super HRP IHC Kit (KIT99-22, MXB, China) was used for immunohistochemistry according to the manufacturer’s instructions. Pancreatic sections were deparaffinized and rehydrated, and citrate buffer (pH 6.0, G1202, Servicebio, China) was used for antigen retrieval. Peroxidase blocking was performed in PBS with 3% H₂O₂ for 10 min, and permeabilization was conducted using 0.2% Triton X-100 (93443, Sigma–Aldrich, U.S.A.) for 45 min at room temperature. The slides were incubated with primary antibodies against IL-1β (1:200, GB11113, Servicebio), nuclear factor κ-B p65 (NF-κB p65) (1:200, GB11042, Servicebio), and Rbpj (1:200, 5313T, Cell Signaling Technology) overnight at 4°C. The slides were incubated with a response enhancer at 37°C for 20 min HRP-conjugated secondary antibody at 37°C for 20 min. AEC (ZLI-0936, ZSGB-Bio, China) or DAB (G1212, Servicebio, China) substrates were used for color development. Five sections in each group and ten 400× images per section were evaluated by calculating the number of positive cells/total number of cells.