Stereological Analysis of Dopaminergic Neurons

Free-floating 35 μm coronal sections containing the SNpc were cut on a horizontal sliding microtome. A total of 8 sections were sampled at 105 µm intervals for each brain region. The free-floating sections were immune-blocked with 4% goat serum in 0.25% Triton/PBS for 2 h and then incubated with Iba-1 antibody (1:2000, Wako Chemicals, Richmond, VA) overnight at 4 °C. On the second day, the sections were washed by 1% BSA in 0.25% Triton/PBS before the incubation with anti-tyrosine hydroxylase (TH) antibody (1:2000, EMD Millipore, Temecula, CA) overnight at 4 °C. The double-label immunofluorescence pictures were taken under the confocal microscope by using Alexa-488 (green) and Alexa-594 (red) conjugated secondary antibodies (1:1000) to visualize the TH immune reactive (THir) or Iba-1 positive cells. Stereological counts of TH+ SNpc neurons were estimated using an optical fractionator method on an Olympus BX50 stereological microscope within user-defined boundaries. Samples were countered in a double-blind manner. Data were expressed as percentage to saline-injected controls [26 (link), 39 (link)].