Anti il 6

Paraffin-embedded sections were incubated at 4 °C with the following primary monoclonal antibodies: anti-IL-1β, anti-IL-6 (Novus Biologicals, Littleton, CO, USA), anti-IL-17, and anti-TNF-α (Abcam, Cambridge, UK). The samples were then incubated with horseradish peroxidase-conjugated secondary antibody for 30 min. The reaction product was developed using 3,3-diaminobenzidine chromogen (Dako, Carpinteria, CA, USA). Histological assessments were conducted by three independent blinded observers. Images were captured using a DP71 digital camera (Olympus, Shinjuku, Tokyo, Japan) attached to a photomicroscope at a magnification of 200 × . Positively stained cells were counted using Adobe Photoshop software (Adobe, San Jose, CA, USA), and the mean values were calculated.

Western blotting was performed according to a standard protocol [34 (link),35 (link)]. Equal amounts of protein (40 μg/well) were subjected to SDS-PAGE (12% gels, Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose membranes. After blocking in TBST (Tris-buffered saline, 0.1% Tween 20) with 5% BSA (Bovine Serum Albumin, Invitrogen, Waltham, MA, USA), membranes were probed overnight at 4 °C with the corresponding primary antibodies, e.g., anti-IL-1β (1:500; Abcam, Cambridge, UK); anti-IL-6 (1:750; Novus Biologicals, Centennial, CO, USA); anti-TNF-α, (1:1000; Invitrogen, Waltham, MA, USA); anti-NF-κB, (1:1000; Invitrogen, Waltham, MA, USA). Anti-actin antibody (1:1000, Santa Cruz, CA, USA) was used for the loading control. After the washing procedure, membranes were incubated with alkaline phosphatase-conjugated secondary antibodies, IgG (1:5000 Santa Cruz, CA, USA) for 1 h at room temperature. The immunoreactive bands were visualized by a colorimetric detection kit (NBT-BCIP; ThermoFisher, Waltham, MA, USA) and protein amounts were analyzed with the ImageJ program (1.46r, NIH, Bethesda, MD, USA).

Paraffin-embedded sections were incubated at 4°C with the following primary monoclonal antibodies: Anti-IL-1β (1:400 dilution, nb600-633, Novus [USA]), anti-MMP-13 (1:300 dilution, ab39012, Abcam [USA]), anti-IL-6 (1:200 dilution, nb600-1131, Novus), anti-RIP1 (1:400 dilution, PA5-20811, Invitrogen [USA]), anti-RIP3 (1:200 dilution, Invitrogen), and anti-phospho-MLKL (1:200 dilution, Abcam). The samples were then incubated with appropriate secondary biotinylated antibodies, followed by 30-min incubation with a streptavidin-peroxidase complex. The reaction product was developed using the 3,3-diaminobenzidine chromogen (Dako, USA).