Primary rat hippocampal neuron culture and immunofluorescent staining

Before the isolation of hippocampi from brains, the neonatal rats (<24 h) were soaked with 75% ethanol for 1–2 min. The hippocampus was then washed and minced in pre-cold D-hanks, and the tissues were digested with 0.25% trypsin supplied with 0.03% EDTA in a 50-mL tube at 37 °C for 20 min. After a 15-min-incubation, the digestion was stopped with the addition of 5% fetal bovine serum (FBS) (Gibco, USA) and the neurons were then centrifuged and suspended in Neurobasal (Gibco) supplied with 10% FBS and filtered with a 74-µM mesh to remove the debris. The suspensions were centrifuged using lymphocyte separation liquid at 2,000 rpm for 20 min. The supernatant was then discarded, and the cells were resuspended with D-hanks. The neuronal suspensions were plated on coverslips coated with 0.01% poly-L-lysine (Sigma, USA) and cultured in Neurobasal containing 10% FBS in a 5% CO₂ incubator at 37 °C. The medium was half replaced every 3–4.5 days.

To identify the isolated primary rat hippocampal neurons, the cells were cultured for 3 days and then fixed with 4% paraformaldehyde for 20 min at room temperature. After being washed three times, the cells were penetrated with 0.1% TritonX-100 (in PBS) for 10 min, followed by blocking with 5% goat serum for 20 min at room temperature. The cells were then incubated with primary antibody mouse anti-Nestin (1:1,000) at 4 °C overnight, followed by 1 h incubation of Cy3-conjugated secondary mouse anti rat NeuN monoclonal antibody (1:500) was incubated at 37 °C for 2 hours and then incubated at 4 °C overnight; biotin labeled Sheep anti mouse IgG (1:200) and avidin anti biotin horseradish peroxidase complex (1:200) were incubated at 37 °C for 2 hours respectively, and each step was 0.01 mmol /L PBS was rinsed for 3 times, and the color was developed by TMB-ST method: distilled water was washed for 3 times; the staining solution was pre incubated at room temperature in dark for 15–20 min; 0.3% H₂O₂ (3.5 µL/mL) was added for 3 times, with an interval of 10 min each time, and the color was controlled under microscope until the positive cells showed clear green; 0.05 m PBS was added into the sections wash for 5 min × 3 times; mount, dry and seal with neutral gum. The images were obtained with Laser scanning confocal microscope (Olympus Fluo View FV1000).