Table 2.

IHC Staining Scheme Design for Immunodetection of Antigens

Section no.	Stained for
1 (most caudal)	Loyez-CFV (high-contrasting histological stain, to confirm presence of PPN using anatomical identifiers)
2	Porin + cholinergic neuron identifier + nuclear marker counterstain
3	Porin + GABAergic neuron identifier + nuclear marker counterstain
4	Porin + glycinergic neuron identifier + nuclear marker counterstain
5	CO-1 (19 kDA) + cholinergic neuron identifier + nuclear marker counterstain
6	CO-1 (19 kDA) + GABAergic neuron identifier + nuclear marker counterstain
7	CO-1 (19 kDA) + glycinergic neuron identifier + nuclear marker counterstain
8	COX/SDH immunoassay
9	IHC for detecting αSYN, including LBs and LNs
10	CO-1 (20 kDA) + Cholinergic neuron identifier + nuclear marker counterstain
11	CO-1 (20 kDA) + GABAergic neuron identifier + nuclear marker counterstain
12	CO-1 (20 kDA) + glycinergic neuron identifier + nuclear marker counterstain
13	COX1 + cholinergic neuron identifier + nuclear marker counterstain
14	COX1 + GABAergic neuron identifier + nuclear marker counterstain
15	COX1 + glycinergic neuron identifier + nuclear marker counterstain
16 (most rostral)	Loyez-CFV high-contrasting histological stain (to confirm presence of PPN) + nuclear marker counterstain

Staining was performed on serial 20-μm thick fresh/frozen sections that followed and contained the PPN. Sections containing the PPN were identified with combined Loyez-CFV IHC, performed at the start of the most caudally available aspect of the serial sections and at the most rostral end, but still within the PPN nucleus. An example of a Loyez-CFV stained section can be seen in Figure 1. In each case where a mitochondrial marker is co-stained with a marker identifying a particular neuronal phenotype (cholinergic, GABAergic, and glycinergic neurons), the mitochondrial protein was detected by using a chromogen (DAB), whereas the neurochemical phenotype was detected by use of a fluorescence-coupled secondary antibody.