4.8. Immunofluorescence Staining and Neurite Network Analysis

Cells were fixed in 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature (RT), followed by blocking with 5% donkey serum in PBS with 0.02% Triton-X100 for 1 h at RT. Then, the cells were incubated with rabbit anti-MAP2 (1:500, Millipore, Billerica, MA, USA), mouse anti-MAP2 (1:500, BD Pharmingen, San Diego, CA, USA), mouse anti-IL-17RA (1:1000, R&D Systems, Minneapolis, MN, USA), mouse anti-IL-17RC (1:500, Abcam, Cambridge, UK), rabbit MHCI (1:500, Abcam), rabbit anti-cleaved caspase 3 (1:500, Cell Signaling Technology), and chicken anti-SMI32 (1:10,000, Covance) at 4°C overnight. Cells were thereafter washed with PBS three times and incubated with Alexa Fluor^® 555 or 647 donkey anti-rabbit IgG or donkey anti-mouse IgG or Alexa Fluor^® 647 (1:500, Abcam) in the dark for 1 h. Finally, the nuclei were stained with Hoechst (Invitrogen). Images were taken using a fluorescent microscope (Zeiss, Goettingen, Germany). In Brief, neurites were recognized by MAP-2 staining; after imaging the neuronal network, the Neuron J plugin (Version 1.52 p) in Image J (ImageJ-win64, open-source) was opened, the image was selected, then we converted the picture into an 8-bit image (Figure S3B). Following this, we labeled all the neurites using the “adding trace” button. After the tracing was completed (Figure S3C), a text file including the neurite length measurement data was generated. We normalized the data within each experiment to the control condition. For each analysis, we took at least 15 pictures at random spots for each condition of every experiment. More than 50 neurons were analyzed in each group, and all experiments were repeated at least three times (see also the Supplementary Materials Table S1).