Rabbit anti nrf2

Immunofluorescence methods have been widely described.
⁴¹ (link) Briefly, 10 μm thick tissue slices were fixed in methanol for 30 min, washed three times in phosphate‐buffered saline (PBS), cleaved with 0.3% Triton X‐100 for 5 min, and mounted in 10% donkey serum working solution (Boster Biological Technology, Wuhan, China) for 60 min. The sliced and mixed samples were incubated with primary antibodies for 12 h at 4°C. Afterward, the samples were washed with PBST to remove residual antibodies and then incubated with the second antibody for 1 h at room temperature in the dark. Primary mouse hippocampal neurons were stained using the ROS red fluorescence probe kit (Biobraille, Shandong, China). The primary antibodies included rabbit anti‐FTMT (1:200; Abcam, USA), mouse anti‐NEUN antibodies (1:100, Abcam, MA, USA), and rabbit anti‐NRF2 (1:300; Affinity, Shanghai, China). Mouse anti‐IBA1 antibodies (1:100; Abcam, MA, USA) and mouse anti‐GFAP antibodies (1:100; Abcam, MA, USA) were used to identify the expression location of FtMt. The secondary antibodies used were donkey anti‐mouse Alexa Fluor 650 (1:100, Boster Biological Technology, Wuhan, China), donkey anti‐rabbit Alexa Fluor 488 (1:100, Boster Biological Technology), and double‐fluorescence immunohistochemical mouse/rabbit kit (ImmunoWay, Beijing, China).

Tissues or cell suspensions were collected for lysis and protein extraction, and the protein concentration was determined by a BCA protein assay kit (Abcam, Shanghai, China). Samples were electrophoresed in 8–12% SDS-PAGE gels, transferred to PVDF membranes (Millipore, Hayward, CA, USA), and then incubated with primary antibodies including rabbit anti-Nrf2 (1:1000, Affinity, Changzhou, China, AF7904), mouse anti-β actin (1:10,000, Affinity, Changzhou, China, T0022), rabbit anti-Bcl-2 (1:1000, Affinity, Changzhou, China, AF6139), rabbit anti-Bax (1:1000, Affinity, Changzhou, China, AF0120), rabbit anti-Cleaved-caspase3 (1:1000, Affinity, Changzhou, China, AF7022), rabbit anti-LC3 (1:1000, Affinity, Changzhou, China, AF5402), rabbit anti-Beclin1 (1:1000, Affinity, Changzhou, China, AF5128), rabbit anti-p62 (1:1000, Affinity, Changzhou, China, AF5384), rabbit anti-NF-κB (1:1000, Affinity, Changzhou, China, AF5006), rabbit anti-PPARγ (1:1000, Affinity, Changzhou, China, AF6284), and rabbit anti-Lamin B (1:10,000, proteintech, Wuhan, China, 12987-1-AP) at 4 °C overnight and HRP-conjugated secondary antibody (proteintech, Wuhan, China, 1:5000) at room temperature for 1 h. Target bands were detected using an enhanced chemiluminescence system, and quantitative analyses were performed using the Image J software (v 1.48, National Institutes of Health, NIH, USA).

After fixation with 4% paraformaldehyde (PFA) for 12–24 h, the brain tissues were embedded in paraffin and sectioned, followed by dewaxing and rehydration. Then, sections were incubated with 0.3% Triton X-100, blocked in 10% goat serum at room temperature for 1 h, and incubated at 4°C overnight with primary antibodies: rabbit anti-Nrf2 (1:500, Affinity), rabbit anti-p62 (1:800, Cell Signaling), mouse anti-Keap1 (1:200, Abcam), rabbit anti-LC3B (1:200, Cell Signaling). Next day, fluorescence-conjugated secondary antibodies that include Alexa Fluor 488 donkey anti-rabbit IgG (1:400) or Alexa Fluor 594 donkey anti-mouse IgG (1:400) were applied to incubate the samples for an hour. The slides were sequentially mounted with DAPI and visualized through fluorescence microscopy.