Analyzing Hippocampal Protein Expression

Western blotting was performed as described previously (Wang et al. 2020 (link)). Total protein from hippocampus was extracted by adding the ice-cold RIPA Lysis Buffer. Proteins were separated by gradient electrophoresis on 10–12.5% SDS-PAGE gels and then transferred onto a polyvinylidene fluoride membrane (Millipore, USA). After they were blocked in 5% skim milk, the membranes were incubated with the following primary antibodies overnight at 4 ℃: claudin 5 (1:1000, Thermo Fisher, USA), occludin (1:10,000, Proteintech, China), Iba1 (1:500, Wako, Japan), and GAPDH (1:10,000, Proteintech, China). After washing with TBST, the corresponding secondary antibodies were applied for 1 h at room temperature (1:10,000, Millipore, USA). Membranes were visualized using ECL solution.