Stereological Analysis of Dopaminergic Neurons

As previously reported (Perez-Pardo et al., 2017 (link)), coronal brain slices of 40 μm were sectioned using a cryostat (CM3050, Leica Microsystems). After incubation with 0.3% H₂O₂ for 30 min and following blocking serum, sections were incubated overnight with rabbit anti-tyrosine hydroxylase (TH; Santa-Cruz Biotechnology, 1:1,000). Next day, sections were incubated with a biotinylated secondary antibody (Jackson ImmunoResearch, 1:200) for 2 h. The avidin-biotin method was used to amplify the signal (ABC Kit, Vector) and staining was visualized using 0.05% DAB solution. Digital images of immunostained sections were captured with an Olympus BX50 microscope equipped with a Leica DFC 320 digital camera. TH-immunopositive neurons were quantified stereologically on regular spaced sections. Analyses were performed by researchers that were blind to the treatment condition of the sample.