Anti glua1

Hippocampal tissue lysates were prepared using radio immune precipitation buffer with protease inhibitors (Cell Biolabs, Inc., San Diego, CA). Protein concentrations were determined with a bicinchoninic acid assay. Proteins were resolved in 10–12% gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). After blocking with 1× RapidBlock solution (Amresco of VWR, Radnor, PA) for 5 min at room temperature, membranes were incubated with the following primary antibodies (Cell Signaling Technology, Danvers, MA) overnight at 4 °C: anti-pS845-GluA1 (1:1,000 dilution), anti-GluA1 (1:1,000) and anti-β-actin (1:1,000). After washing, the membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000) in 1× RapidBlock solution for 1 h at room temperature and visualised using an enhanced chemiluminescence detection system (Millipore, Bedford, MA). The optical density of immunoreactive bands was measured using ImageJ software (National Institutes of Health, Bethesda, MD). The results were normalised to the quantity of β-actin in each sample lane.

Immunofluorescence was performed following previous studies in which rats anesthetized to death were immobilized by heart perfusion with 0.9% saline and 4% paraformaldehyde [33 (link)]. After repeated perfusion to the anatomy of the brain tissue after the rat body stiffened, the prefrontal regions, such as the coronal section, in turn in 20%, 25%, and 30% sucrose-fixed dehydration, gel embedding, freezing, the parts with cryostat coronary frozen section (10 microns), membrane breaking, repair, washing with phosphate-buffered saline (PBS) 3 times and sealed with 5% sheep serum. The slices were incubated for 12 h in anti-GluA1 (1:200, Cell Signaling, #13185, Shanghai, China), anti-BDNF (1:200, Abcam, ab108319, Shanghai, China), and anti-PSD-95 (1:200, Cell Signaling, #3450, Shanghai, China) at 4°, then incubated at constant temperature in the second antibody for an hour. Finally, the nuclei were stained with DAPI (Solarbio, C0065, Beijing, China), and the immunofluorescence of the treated specimens was observed by fluorescence microscope (Olympus Corporation, Japan). The secondary antibody used was Alexa Fluor 488-conjugated Goat Anti-Rabbit IgG (1:200, Proteintech, SA00006-2, Wuhan, China) and Alexa Fluor 594-conjugated Affinipure Goat Anti-Mouse IgG (1:200, Proteintech, SA00006-3, Wuhan, China).