Nf κb p65

Antibodies were used to evaluate colon expression of tumor necrosis factor alpha (TNF-α) (ABclonal Technology, MA, USA, catalog number: A0277), nuclear factor kappa beta p65 (NF-κB p65) (Bioss antibodies, MA, USA, catalog number: bs-20159R), and COX-2 (ABclonal Technology, MA, USA, catalog number: A1253), using Avidin–Biotin Complex (ABC), immunohistochemical staining protocol, according to the manufacturer's instructions.
Sections (4 μm) of colons were incubated for 20 min in 0.3% H2O2 to block the endogenous peroxidase activity. The section slides were incubated in a 100 °C water bath for 10 min in 0.01 M PBS buffer solution, then blocked with 2% BSA for 30 min. Then, the sections were incubated with the anti-rat antibodies overnight at 4 °C. The slides were washed and then incubated with HRP secondary antibody at 37 °C for 1 h. The sections were subsequently treated with DAB working solution for 4 min and finally counterstained with Mayer’s hematoxylin. Tissue sections were observed, photographed with a microscope (400 × magnification) and were semi-quantified.

OA‐related protein expression in chondrocytes was detected by Western blotting. Briefly, protein extracted from chondrocytes were separated by SDS‐PAGE and transferred to PVDF membrane. Membranes were blotted with primary antibodies recognizing TLR‐2 (Abcam, USA), NF‐κB p65 (Bioss, China), NF‐κB phospho‐p65 (Bioss), and GAPDH (Bioss), respectively. All blots were probed with horseradish peroxidase (HRP)‐conjugated secondary antibodies (Zen Bioscience, China) and revealed using the enhanced chemiluminescence (Millipore, USA) detection system.