Rabbit anti nrf2 antibody

IEC-6 cells (2 × 10⁵ cells/well) were seeded on coverslips in a 12-well plate and treated with NIV and DON (5 μM) for 1 h, alone or in a mixture, or in the presence of LPS + IFN for 24 h for nitrotyrosine formation and for 1 h for Nuclear Factor-kB (NF-κB ) and Nuclear Factor (erythroid-derived 2)-like 2 (Nrf2) activation. In some experiments, DPI (10 μM) was added 1 h before NIV and DON incubation. After, the treatment cells were fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% saponin in PBS. After blocking with bovine serum albumin (BSA) and PBS, cells were incubated with rabbit anti-nitrotyrosine (Millipore, Billerica, MA, USA), rabbit anti-Nrf2 antibody (Santa Cruz Biotechnologies, Dallas, TX, USA), or rabbit anti-phospho p65 NF-κB (Santa Cruz Biotechnologies, Dallas, TX, USA), for 1 h at 37 °C. The slides were then washed three times with PBS and fluorescein-conjugated secondary antibody (FITC) was added for 1 h. 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) was used for the counterstaining of nuclei. Lastly, coverslips were mounted in mounting medium and fluorescent images were taken under the Laser Confocal Microscope (Leica TCS SP5, Leica, Wetzalar, Germany) as previously reported [27 (link)].

The kidneys were fixed with 10% formalin and embedded in paraffin. Kidney sections (4 μm in thickness) were incubated in 3% H₂O₂ for 15 min at room temperature to block endogenous peroxidase activity. After boiling in antigen retrieval solution (1 mmol/L Tris–HCl, 0.1 mmol/L EDTA, pH 8.0) for 15 min at high power in a microwave oven, the sections were incubated overnight at 4°C with a rabbit anti-Nrf2 antibody (Santa Cruz, Dallas, TX, United States). After washing with PBS, the secondary antibody was applied, and the signal was visualized using an ABC kit (Santa Cruz, Dallas, TX, United States).