Bs 8845r

Hippocampal tissues were homogenized in RIPA cell lysate (Beyotime, P0013B), centrifuged at 12,000 × g for 15 min, and the supernatant was collected. Each protein sample was loaded into the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel spiked wells. Electrophoresis was performed at constant pressure of 80 V for approximately 1 h. The proteins were transferred to a polyvinylidene fluoride membrane (Millipore, IPVH00010), fixed with western closure solution (5% skim milk powder), slowly shaken on a shaker for 2 h at room temperature, and then incubated with the following primary antibodies: rabbit anti-postsynaptic density-95 (PSD-95) antibody (1:2000, abcam, ab238135), rabbit anti-synaptophysin (SYN) antibody (1:1000, Bioss, bs-8845R), rabbit anti-BDNF antibody (1:1000, abcam, ab108319), and rabbit tyrosine kinase receptor B (TrkB) antibody (1:5000, abcam, ab187041). The samples were further incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:20000, Zsbio, ZB-2301) for 1.2 h. The membranes were washed with phosphate-buffered saline with Tween and the ECL luminescence kit (Thermo, 340,958) was used to detect the proteins. Finally, the intensity of the bands was analyzed by Image J software (Media Cybernetics, United States).

The RIPA cell lysis solution (Beyotime, P0013B) was added to the hippocampal tissue for lysis. It was then centrifuged at 12,000 × g for 15 min, after which the supernatant was carefully collected. Protein samples were loaded into SDS–PAGE gel spiked wells. Following constant pressure 80 v electrophoresis for 1 h, samples were transferred to a PVDF membrane (Millipore, IPVH00010), specific transfer times were 60 min for PSD‐95, 35 min for SYN, 20 min for BDNF, and 65 min for TrkB. After the film transfer, western blocking solution (5% skim milk powder) was added and blocked for 2 h at room temperature. Then, the membranes were incubated with diluted rabbit antibodies against PSD‐95 (1:2000, Abcam, ab238135), SYN (1:1000, Bioss, bs‐8845R), BDNF (1:1000, Abcam, ab108319), and TrkB (1:5000, Abcam, ab187041) overnight at 4°C. Membranes were then incubated for 1.2 h at room temperature with a horseradish peroxidase‐conjugated goat anti‐rabbit immunoglobulin G (1:20000, Zsbio, ZB‐2301). Proteins were detected using an ECL ultrasensitive luminescence kit (Thermo, 340958) according to the manufacturer's instructions. ImageJ software was used for bands analysis (Media Cybernetics).