DA neurons were fixed for 15 min in 4% paraformaldehyde (Electron Microscopy Sciences) in PBS at room temperature and then incubated in blocking buffer (1X PBS, 1% Goat serum, 4% BSA) for 1 hr. Cells were permeabilized in 0.2% Triton X-100 in PBS for 10 minutes and incubated overnight with primary antibodies. Primary antibodies are listed in the Supplemental Methods. The Click-iT EdU Alexa Fluor® 488 Imaging Kit was used to assess the mitotic state of TH+ neurons. Confocal and spinning disk epifluorescence confocal imaging analysis was performed using a Zeiss LSM700 or Olympus Axiovert 100 M confocal microscope (Carl Zeiss, Olympus). Cells were counted using Zeiss confocal microscope software (Zeiss). Ten images from one coverslip from two independent experiments were used for counting.
