Stereological Quantification of Dopaminergic Neurons

Stereology was initially performed on serial sections from the midbrain following a protocol adapted from Tapias et al. 2014^{67 (link)}. Briefly, tissue was immunolabeled for TH, MAP2, and DAPI and imaged at ×20 on a Nikon90i fluorescent microscope in the Center for Biologic Imaging at the University of Pittsburgh. Images were analyzed in Nikon Elements software counting the number of dopaminergic neurons determined by overlap between DAPI, MAP2, and TH within a region of interest defining the substantia nigra pars compacta as previously described^{67 (link)}. Subsequent experiments were performed with a modified method of stereology utilizing overlap between TH and fluorescent Nissl (NeuroTrace 647, Life Technologies) within the substantia nigra pars compacta as previously described^{68 (link),69} . These images were obtained on an Olympus BX61VS slide scanning microscope, and analysis was performed utilizing Nikon Elements software.