2.6. Immunofluorescent Staining and Measurement of Neurite Outgrowth

The cells cultured on coverslips were washed with phosphate-buffered saline (PBS, Invitrogen) and fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 minutes at room temperature. After three rinses (10 minutes each) with PBS containing Tween 20 (Invitrogen) (PBST), the cells were incubated in blocking solution consisting of PBST and 10% BSA (Sigma-Aldrich) for 30 minutes at room temperature. Subsequently, the samples were hybridized with the primary anti-TH (1 : 500, Millipore) and TUBB3 (1 : 500, Biolegend) antibodies in blocking solution overnight at 4°C. After three rinses (10 minutes each) with PBST, the cells were incubated with the diluted secondary antibody conjugated with Alexa 594 or Alexa 647 (Thermo Fisher Scientific) in blocking solution in the dark for 1 hour. The cells were counterstained with 4′-6-diamidino-2-phenylindole (DAPI, 1 : 1 000; Thermo Fisher Scientific) for nuclear detection. Subsequently, the coverslips were mounted with DAKO mounting solution onto microscopic slides. The cells were observed using a Leica TCS confocal microscope. The neurite outgrowth features (TH-positive) including total outgrowth, processes, and branches were counted randomly with more than 500 cells and assessed by MetaMorph microscopy automation and using the image analysis software (Molecular Devices).