Quantification of Dopaminergic Neurons in Mouse Brain

Immunostaining was performed as described previously^{24 (link)}. Mouse brains were cut into 35 µm sections on a horizontal sliding microtome. The free-floating sections were immune-blocked with 4% goat serum in 0.25% triton/PBS for 1 h. Dopaminergic neurons were stained with anti-tyrosine hydroxylase (TH, 1:4,000, overnight at 4 °C). Sections were incubated with biotinylated secondary antibody for 1 h followed by incubation with Vectastain ABC reagents (Vector Labs, Burlingame, CA) for 40 min and then color was developed with 3,3-diaminobenzidine. To monitor DA neuro degeneration, two individuals blind to the treatment counted the number of TH-immunoreactive (TH-IR) neurons in the SN pars compacta (SNpc) of six evenly spaced brain sections from a series of 24 sections that covered the entire SN^{26 (link)}. Stereological counts of THir SNpc neurons were estimated using an optical fractionator method on an Olympus BX50 stereological microscope within user-defined boundaries^{24 (link)}. All immunohistochemistry images were captured by a Leica Aperio AT2 Scanner.