Anti psd95

On D4, D5, and D6, the cells were washed with ice-cold PBS (Invitrogen). Cell lysates were prepared using RIPA buffer, as previously described [30 (link)]. Equal volumes of cell lysates were applied to SDS-PAGE, and transferred to PVDF for immunoblotting [30 (link)]. The following antibodies were used at 1/1000 dilution: anti-synaptophysin (Cell Signaling 4329S), anti-SNAP25 (Santa Cruz sc-7538), anti-PSD95 (Cell Signaling, 2507S), anti-GAP43 (Cell signaling, 8945S), and anti-β-tubulin (Sigma T5201). Secondary antibodies, anti-mouse HRP-linked, and anti-rabbit HRP linked from Cell Signaling and anti-goat HRP-linked from Santa Cruz were used to detect the bands with Supersignal chemiluminescence ECL kit (Millipore, Molsheim, France). Gel images were obtained while using the Fusion Imaging System (Fusion Fx5; Vilber Lourmat, France), and densitometric analysis was performed using the freeware ImageJ.

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).

Samples were lysed in TEN buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, and 1% NP-40, supplemented with protease inhibitors and phosphatase inhibitors. Protein concentration was determined by BCA assay (BCA Protein Assay Kit, Thermo Fisher Scientific). Equal amounts of protein samples were subjected to SDS-polyacrylamide gel electrophoresis and PVDF membrane transfer. Proteins were identified by incubating with indicated primary antibodies and then with appropriate HRP-conjugated secondary antibodies. Protein band intensities were determined using the Image J software [45 (link)]. Antibodies used were: anti-GAPDH (Abways, ab0037), anti-PSD93 (Abcam, ab151721), anti-PSD95 (Cell Signaling Technology, 3450 S), anti-Akt (Cell Signaling Technology, 9272 S), anti-phosphorylated Akt (Cell Signaling Technology, 9271 S), and HRP-conjugated secondary antibodies (Thermo Fisher Scientific, 31430 and 31460).