Take 8 cryosections cut at 40μm to equally sample the substantia nigra, pars compacta (SNc) and perform diaminobenzidine staining with a tyrosine hydroxylase antibody.

Note: I take every 6th section from Bregma -2.54mm to -3.88mm

Use StereoInvestigator software to count all TH+ cells. For this, randomly sample the SNc with a 150μm x 150μm grid and apply an optical fractionator consisting of a 75μm x 75μm square. Calculate the mean section thickness during counting with a frequency of 10 frames (~3 measurements per hemisphere).