Anti tnf α

The invasive potential of HTR8/SVneo cells was assessed in vitro using a BD BioCoat Matrigel Invasion Chamber (BD Biosciences, Bedford, USA) as previously described (22 (link)) with modifications. Briefly, culture medium was added into a 24-well plate and transwell inserts were plated into the wells for 2 h rehydration at 37°C. Culture medium (700 μl, 1640 with 10% FBS) was added to the lower chamber of all the wells with the transwell insert. Next, cells (4 ×10⁴ cells/well) suspended in 50 μl of RPMI 1640 with 1% FBS were combined with 50 μl specific dMϕ-CM sample, including FC-CM, Mock, Control dMϕ-CM, PRL dMϕ-CM, Control dMϕ-CM plus 10 ng/ml TNFα (Peprotech, USA), PRL dMϕ-CM plus 20 µg/ml anti-TNFα (Proteintech, USA), Mock plus 20 µg/ml anti-TNFα (Proteintech), siMNSFβ-Mϕ CM, NC-Mϕ CM, and siMNSFβ-Mϕ CM plus 10 ng/ml TNFα (Peprotech, USA), and the cell solutions were seeded in the upper chamber over the Matrigel matrix in a total volume of 100 μl. After incubating for 28 h, the noninvaded cells on the upper surface of the membrane were removed using a cotton swab. Membranes were then fixed with 4% paraformaldehyde, stained with 0.1% crystal violet (Sangon Biotech, Co., Ltd. Shanghai, China), and washed with ddH₂O. The stained cells were counted by ImageJ software.

Kidney sections (4 μm) were first heated in 60°C, then deparaffinized in xylene, rehydrated in graded alcohol, and treated with 0.01 M sodium citrate buffer and 3% H₂O₂ to block endogenous peroxidase activity (immunofluorescence did not require this step). Then, 10% goat serum was added to block nonspecific antigen binding for 40 min in 37°C and in next step kidney sections were incubated with primary antibodies like anti-KIM-1 (1 : 200, Bioss), anti-CD68 (1 : 200, Proteintech), anti-TNF-α (1 : 200, Proteintech), anti-4-HNE (1 : 50, Abcam), and anti-Ly6G (1 : 100, Abcam) overnight at 4°C. Samples were then incubated in secondary antibodies (HRP conjugated for IHC and fluorescently labeled for IF) for 1 h at 37°C. The TdT-mediated dUTP nick-end labeling (TUNEL) staining was performed using commercial kits according to the manufacturer's instructions (Beyotime Biotechnology, Shanghai, China). DCF, DHE, and JC-1 staining was performed using commercial kits following the manufacturer's instructions (Beyotime Biotechnology, Shanghai, China). Slides were imaged using a microscope (Leica, Germany) or a confocal laser scanning microscope (Zeiss, Germany).