Anti tnf α

Fixed joint tissues embedded in paraffin were cut into 7-µm-thick sections, dewaxed using xylene, dehydrated through an alcohol series, and stained with hematoxylin and eosin, safranin O, or toluidine blue to detect proteoglycans. Endogenous peroxidase activity was quenched with methanol/3% H₂O₂. Immunohistochemistry was performed using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA). Sections were incubated with specific antibodies (i.e., anti-TNFα, anti-IL-1β, anti-IL-6, anti-IL-17, anti-RANKL, anti-VEGF, and anti-HIF-1α; all from R&D Systems) overnight at 4 °C. The tissue sections were then incubated with a biotinylated secondary antibody, followed by a streptavidin-peroxidase complex for 1 h. The final color product was developed using diaminobenzidine as the chromogen (DAKO, Carpinteria, CA, USA).

S. aureus supernatant was obtained by centrifugation and filtration. HKLAC was obtained by incubating LAC cells in PBS (1 × 10⁹ CFU/mL) at 65 °C for 2 h to inactivate the bacteria. Neutrophil lysates were run on bolt 4–12% Bis-Tris Plus gels (Life Technologies) and transferred to polyvinylidene difluoride membranes (Millipore). Then the membrane was blocked with 5% milk in TBST (Tris-buffered saline plus Tween) for 1.5 h at room temperature. Immunodetection was performed using anti-phospho-MLKL (Ser358) (Abcam, Cambridge, UK), anti-MLKL (Abcam, Cambridge, UK), anti-TNFα (R&D Systems, USA), and β-actin (Sigma Aldrich Chemical Co, USA) antibodies followed by secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology Inc, USA).

Protein extracts were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes (Millipore, Germany). Protein expression was detected by western blot analysis. Antibodies used herein including anti-IL-6, anti-TNF-α, anti-p-AKT, anti-AKT, anti-SRC, anti-p-SRC, anti-VEGFA, anti-MAPK1, anti-IL-1β, anti-EGFR and β-actin were obtained from R&D Systems. Band density was quantified using ImageJ software and normalised to the corresponding control group.

Ham’s F12 medium was obtained from GE Healthcare Europe (Freiburg, Germany). RPMI-1640, GlutaMax and FCS were purchased from Life Technologies (Darmstadt, Germany). PBS was acquired from Biochrom (Berlin, Germany). Pargyline (Parg), phorbol 12-myristate 13-acetate (PMA) and trichostatin A (TSA) were supplied by Sigma-Aldrich Chemie (Munich, Germany). IL-1β, IL-1ra, TNF-α and anti-TNF-α were obtained from R&D Systems (Wiesbaden, Germany). Flagellin from Salmonella typhimurium was purchased from InvivoGen (San Diego, USA) and actinomycin D was supplied from Biovision (Milpitas, USA). All other applied chemicals were of analytical grade and acquired from commercial sources.

Paraffin-embedded sections, 3-4 μm thick, were used for routine H&E staining and for immunoperoxidase immunohistochemical examination with the DAKO EnVision detection system. The following primary monoclonal antibodies were used: anti-TNFα (R&D, UK) and matrix metalloproteinase 9 (MMP9) (Novocastra).
For immunohistochemistry, the paraffin-embedded sections were placed on adhesive plates and dried at 56°C for 24 hours and were later deparaffinated in a series of xylenes and alcohols with decreasing concentrations. The activity of endogenous peroxidase was inhibited with 3% hydrogen peroxide solution in methanol for 5 minutes.
In order to retrieve the antigenicity of tissues and allow them to react with antibodies, specific procedures were used for each tested antibody, according to the manufacturer's instructions. After incubation with diluted antibodies for 60 minutes at room temperature, they were washed with Tris buffer twice. DAKO EnVision double-step visualization system was then applied in order to visualize the antigen-antibody reaction. In the case of a positive immunohistochemical reaction, cellular nuclei were stained with Meyer haematoxylin for 2 minutes. After dehydration and processing through a series of acetones and xylenes, the sections were fixed in Canadian balm.