Unbiased Stereological Assessment of Microglia

The fractionator method of unbiased stereology was utilized to evaluate microglial activation in the SNpc, as previously described [7 (link), 46 (link)]. Briefly, TH-stained coronal sections located at − 3.14, − 3.26, and − 3.38 mm bregma were used to delineate the SNpc. A 40× objective was used to score stages of the changes in microglial morphology of IBA-1 stained microglia within the SNpc. Morphological parameters were used to identify four distinct morphology classifications (0–3) as previously described [7 (link), 46 (link)]. Samples were counted in a blind manner by two individuals using an Olympus BX51 microscope (Center Valley, PA) and newCAST software (Visiopharm, Hoersholm, Denmark). Conclusions were drawn only when differences in counts were less than 12% between individuals.