Immunofluorescence Staining of Neurons

Immunofluorescence was performed as previously described (Xie et al., 2018 (link)). Briefly, cells were fixed with 4% paraformaldehyde at 4°C for 20 min, permeabilized with 0.5% Triton X‐100 in PBS, and incubated at 4°C with antibodies against NeuN (cat. no. ab104224, Abcam, diluted with 5%BSA to 1:250) and MAP2 (cat. no. ab183830, Abcam, diluted with 5% BSA to 1:250) overnight. Cells were then incubated with Goat anti‐Rabbit IgG (H+L) Secondary Antibody (FITC; cat. no. 31635, diluted with 5%BSA to 1:250) and Goat anti‐Rabbit IgG (H+L) Cross‐Adsorbed Secondary Antibody (Alexa Fluor™ 546; cat. no. A‐11010, diluted with 5% BSA to 1:250, Thermo Fisher Scientific, Inc.) at 37°C for 1 h. Nuclei were stained with DAPI for 5 min and viewed using an LSM780 laser scanning confocal microscope (scale bar = 50 μm)(Carl Zeiss GmbH).
To detect early apoptotic cells, treated neurons were stained with Annexin V‐FITC (Beyotime Institute of Biotechnology) in PBS for 15 min at 37°C, washed with PBS, and incubated with Hoechst 33342 (Beyotime Institute of Biotechnology) to stain the nuclei for 5 min (room temperature ), washed with PBS, and viewed using an LSM780 laser scanning confocal microscope (scale bar = 50 μm).