2.4. Fluorescent labelling
2.4.1. I mmunofluorescent staining
For immunofluorescent staining, each coverslip with neurons was taken, cleaned with PBS (pH=7.4) 3 times, fixed with 4% paraformaldehyde at room temperature for 10 min, then rinsed with PBS once. The fixed neurons were dipped in 0.1% triton for 10 min, then blocked with 3% serum albumin for 30 min after PBS washing. Removing the blocking solution, samples were covered with the axonal tubulin antibody (1:1000 diluted with PBS) that specifically labels β-tubulin III, standing at 37℃ for 2 h. Then, the secondary antibody labelled with Alexa Fluor®488 replaced the primary antibody, incubated at 37℃ for 3 h, followed by 3 times wash, stained with 100 μL Hoechst solution for 5 min. Finally, after washing with PBS, stained neurons were optically imaged under a multichannel microscope (Suzhou NIR-Optics Technology Co., Ltd., China).