Immunofluorescence Staining of Neuronal Cells

Cells were immobilized with 4% precooling paraformaldehyde (P1110; Solarbio, Beijing, China) for 30 min and permeabilized with 0.3% Triton X-100 (T8200; Solarbio) at room temperature for 10 min. After being blocked in 1% bovine serum albumin (BSA; A8020; Solarbio) for 30 min, cells were incubated with primary antibodies including anti-βIII tubulin (1 μg/mL) and anti-CNPase (5 µg/mL) at 4℃ overnight. Post three times of washing in PBS (806552; Sigma-Aldrich), the primary antibodies were identified with Alexa Fluor 594 goat-anti rabbit antibodies (B40925; Invitrogen) for 60-min incubation at room temperature. Subsequently, the nuclei were counter-stained with DAPI (C1002; Beyotime) at 37℃ for 10 min. Ultimately, the slides were mounted and observed under a fluorescence microscope (Leica, TCS SP5II, Germany).