Rabbit anti nrf2 antibody

Nuclear NRF2 translocation was determined by the immunofluorescence assay as previously described by Zhang et al. [35 (link)], with minor modifications. Briefly, IPEC-1 cells were fixed in 4% paraformaldehyde solution in 0.1 M PBS at room temperature for 30 min after washing twice with PBS. The fixed cells were further permeabilized with 0.5% Triton X-100 for an additional 30 min. Following the washing with PBS, cells were incubated with the blocking buffer (5% bovine serum albumin in PBS) for 1 h at 4 °C before incubation with a rabbit anti-NRF2 antibody (1: 100, Proteintech Group, Inc., Chicago, IL, USA) in PBS at 4 °C overnight. After washing with PBS with 0.1% Tween 20, cells were incubated with a secondary antibody, Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin G (1: 100, Invitrogen, Carlsbad, CA, USA), in dark conditions for 1 h at 4 °C. The 5 µg/mL 4,6-diamidino-2-phenylindole was adopted for nuclear staining. Images were visualized with an Eclipse 80i inverted fluorescence microscope (Nikon Instruments, Tokyo, Japan), and nuclear NRF2 fluorescence intensity was calculated using the Image J software (National Institute of Health, Bethesda, MD, USA) and normalized against control cells.

For IHC, 3 μm thick paraffin-embedded renal sections were deparaffinized, hydrated and blocked, following incubation with primary antibodies including rabbit anti-Nrf2 antibody (catalog number: 16396-1-AP, Proteintech, Chicago, IL, USA) and anti-Keap1 antibody (catalog number: sc-365626, Santa Cruz, Dallas, TX, USA) overnight at 4 °C. Following diamino-benzidine (DAB) reaction, the sections were then incubated in horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA) for 30 min. Sections were photographed under a light microscope (Olympus CX23, Tokyo, Japan) and quantified with image-pro plus 6.0 software (Media Cybernetics, Rockville, MD, USA) as previously reported [62 ]. After intensity calibration, positive regions were extracted and separated. The positive color segmentation threshold was based on the fixed threshold value of hue, saturation, and intensity (HSI). Images segmentation and area measurement was based on the same HSI profile for all images. Integral optical density (IOD) and the area were measured and the protein expression levels were analyzed by calculating IOD/Area. Four fields of view were randomly selected from each sample.

Two livers from each group were combined into one sample and total protein was extracted for western blot analysis (one sample mixed with liver tissues from two fish, n = 8).
Western blotting was performed as described previously [83 (link)]. The antibodies used were as follows: rabbit anti-β-actin antibody (1:2000, Proteintech, Wuhan, China), rabbit anti-α-SMA antibody (1:1000, Proteintech, Wuhan, China), rabbit anti-SIRT1 (1:1500, Proteintech, Wuhan, China), rabbit anti-NOX4 antibody (1:1000, Proteintech, Wuhan, China), rabbit anti-NRF2 antibody (1:1500, Proteintech, Wuhan, China), rabbit anti-phospho-AKT (Ser473) antibody (1:2000, Cell Signaling), rabbit anti-phospho-AMPK (Thr172) antibody (1:1000, Cell Signaling), rabbit anti-AMPK antibody (1:1000, Proteintech, Wuhan, China). Protein expression was normalized to that of β-actin.