Neurons were fixed for 20 minutes in 4% paraformaldehyde in PBS, washed twice with PBS and permeabilized with 0.3% Triton X-100 in PBS for 10 minutes. Cells were blocked in 1-2% BSA, 5% normal goat serum in 0.3% Triton X-100/PBS for 1 hour at room temperature. Neurons were incubated with the following primary antibodies: anti β -III-tubulin (Covance # MMS-435P, 1:1000 or Covance # MRB-435P, 1:1000), TOM20 (BD Bioscience # 612278, 1:300), Tyrosine Hydroxylase (Merck-millipore # 657012, 1:1000), tau (DAKO # A002401-2, 1:300), MAP2 (Sigma # M4403, 1:300). Primary antibodies were incubated overnight, washed in 0.3% Triton X-100/PBS and then incubated with Alexa-conjugated anti-rabbit or anti-mouse antibodies at 1:500 for 1 hour at room temperature. Micropatterned substrates were mounted in DAPI Fluoromount (Southern Biotech).
