Ab225942

Total protein of the cortex of the ischemia side in mice brains or cultured cells was extracted using a RIPA lysis buffer (Beyotime Institute of Biotechnology, China). Approximately equal amounts of protein were separated by 12% SDS-PAGE and transferred onto PVDF membranes. After blocking in 5% skim milk, the membranes were incubated with primary antibodies, including FGF19 (1:500, ab225942, Abcam), GSK-3β (1:500, ab93926, Abcam), phosphorylated-GSK-3β (1:500, ab75814, Abcam), Nrf2 (1:500, ab62352, Abcam), Lamin B2 (1:1000, ab8983, Abcam) and GAPDH (1:2000, ab8425, Abcam), overnight at 4 °C. Afterwards, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000, ab205718, Abcam) at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescent (ECL) kit.

The mice brain tissues were fixed and cut coronally into 5 mm-thick serial sections, and then immunohistochemistry was performed with an immunohistochemical kit (Boster Biological Technology, Wuhan, China) using FGF19 (1:500, ab225942, Abcam) antibody and cleaved caspase-3 (1:300; ab2302, Abcam) antibody according to the manufacturer’s instructions. A blinded investigator used a microscope to take images and chose images randomly for each section. Analysis of FGF19 expression was conducted using Image-Pro Plus software.