Double Immunofluorescence Staining
In order to obtain histological results, mice were anesthetized with 1%
barbital sodium (100 mg/kg) (Aladdin, China) before being transcranially
perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by
4% paraformaldehyde (PFA). After collecting the brains, they were fixed
in 4% PFA at 4 °C for 24 hours and cryopreserved in 30% sucrose until
sectioning. A sliding microtome (Leica, Germany) was used to section the
brain coronally into 35 m sections containing the hippocampal area which
is about 2 mm from the Bregma. Then the brain sections from hippocampus
were blocked for 10 min in PBS containing 10% bovine serum albumin
(BSA) and then incubated overnight at 4 °C with the primary antibodies
NeuN (Mouse monoclonal) (1:100, Abcam, USA) and synaptophysin (Rabbit
monoclonal) (1:100, CST, USA) or PSD95 (Rabbit monoclonal) (1:100, CST,
USA). Subsequently, the sections were incubated with the secondary
antibodies [Alexa Fluor 555-conjugated anti-mouse (1:1000 dilution,
Thermo Fisher Scientific, USA), Alexa Fluor 488-conjugated anti-rabbit
(1:1000 dilution, Thermo Fisher Scientific, USA)] for 2h at room
temperature. Each of the steps above was followed by three rinses in
PBS lasting 10 minutes each. At the end the sections were mounted by
fluorescent mounting medium (Sigma, USA). Laser-scanning confocal
microscopes (Leica TCS SP2, Germany) were used to capture the confocal
images.