Anti cleaved caspase 3

For immunohistochemical analysis, sections were deparaffinized through xylene and ethanol series. All sections were boiled in 10 mmol/l sodium citrate antigen retrieval buffer at 95°C for 20 min and then washed three times with PBS. The sections were exposed to 3% hydrogen peroxide for 15 min to quench endogenous peroxidase activity and then washed three times with PBS. Next, the sections were blocked with 10% goat serum for 1 h and then incubated overnight at 4°C with anti-Bax (1:200 dilution; Bioworld, Minnesota, United States), anti–Bcl-2 (1:200 dilution; Bioworld), anti-cleaved caspase 3 (1:200 dilution; Bioworld), anti-zonula occludens-1 (ZO-1; 1:150 dilution; Bioworld), anti-occludin (1:200 dilution; Abcam), anti-claudin 1 (1:200 dilution; Abcam), and anti-AChE (1:200 dilution; Proteintech, Wuhan, China) antibodies. After three washes with PBS, the sections were incubated with secondary antibodies for 30 min at 37°C and then washed three times with PBS. Diaminobenzidine was used to develop the antibody staining, and hematoxylin counterstaining was used to visualize the nuclei. Images were obtained under a light microscope (BX53, Olympus, Japan) and analyzed using Image-Pro Plus 6.0 (Media Cybernetics).

Western blot (WB) analysis was performed using the standard methods previously described to detect protein levels of apoptosis, autophagy, and endoplasmic reticulum-associated proteins in each group of brains (Gao et al., 2017b (link)). In brief, the supernatant containing the protein was extracted after being lysed the brain tissue, the protein concentration was adjusted, and the same amount of protein was separated by gel electrophoresis and transferred to a Hybond-polyvinylidene difluoride (PVDF) membrane. The PVDF membrane was then incubated with the primary antibodies to anti-NF-κB (1:500, CST), anti-ST2 (1:500, Abcam, ab25877), anti-IL-33 (1:500, R&D, AF3626), anti-Beclin-1 (1:1000, Bioworld), anti-LC3B (1:3000, Abcam), anti-P62 (1:1000, Abcam), anti-Bcl-2 (1:500, Abcam), anti-cleaved-caspase-3 (1:500, Bioworld) and anti-β-actin (1:10,000, Sigma). anti-β-actin was used as a loading control. Then, the PVDF was incubated with the respective HRP-conjugated secondary antibody for 2 h at room temperature. Blots were detected with the ECL chemiluminescence system (Beyotime Institute of Biotechnology) and were captured on autoradiographic films (Kodak). Films were scanned and densitometric analysis of the bands was performed with Sigma Scan Pro 5.