Ab167161

RIPA lysis buffer, containing 50 mM Tris, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, 1.5 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride, was used to homogenize hippocampal tissues. The lysate was centrifuged for 20 min at 12,000×g and 4 °C. The protein concentration was determined using a BCA assay (Thermo Scientific), and 30 μg of protein was loaded into each lane of a modified gel for analysis by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), which were then blocked for 1 h with 5% nonfat milk in Tris-buffered saline Tween 20. The blocked membranes were probed overnight with specific primary antibodies diluted in 5% nonfat milk, as instructed by the manufacturer. Rabbit anti-APP (Abcam, ab15372, 1: 500), rabbit anti-occludin (Abcam, ab167161, 1:500), and mouse anti-GAPDH (Proteintech, 1:1,000) were the primary antibodies used. Before exposure to film, the membranes were washed, incubated with secondary antibodies (1:1,000), and then washed and treated with ECL reagent. Image Lab software (Bio-Rad, Richmond, CA, USA) was used to perform the densitometry analysis, and ImageJ was employed to calculate the results.

Immunostaining was performed in accordance with previously published protocols [58 (link)] using primary antibodies for ZO-1 (1:500, Abcam, ab216880), occludin (1:500, Abcam, ab167161), and Nrf2 (1:500, Proteintech, Wuhan, China, 16396-1-AP). After incubation with the corresponding secondary antibody (1:1000), cell nuclei were stained with DAPI (Roche, Shanghai, China) for 15 min, after which fluorescence microscopy was used for analysis and image acquisition (Olympus, Tokyo, Japan).