Histological methods

The process leading to the choice and evaluation of immunohistochemical methods for demonstrating pathological α-synuclein has been described in previous publications [16, 24, 67-69]. The standard method used at AZSAND employs proteinase K pretreatment, which not only results in superior epitope exposure but also may assist with the pathological specificity of the stain by digesting normal, non-aggregated α-synuclein, which is abundant in all nervous tissue. Using an antibody specific for α-synuclein phosphorylated at serine 129 (pSyn) [70-74] also helps identify stained structures as pathological since normal control subjects do not have pSyn-immunoreactive brain tissue elements [4,75]. Complete details of the staining procedure have been previously described [76] and so only a brief description is given here.

From each postmortem subject, three sections from vagus nerve and three sections of stomach were stained and examined. Formalin-fixed, paraffin-embedded 5-7 μm sections were deparaffinized and treated with 1:100 proteinase K (Enzo Life Sciences, Farmingdale, NY) at 37° C for 20 minutes, followed by suppression of endogenous peroxidase activity with 1% hydrogen peroxide for 30 minutes, incubation in primary antibody against α-synuclein phosphorylated at serine 129, diluted 1:10,000 [71-74], incubation in biotinylated secondary antibody, avidin-biotin peroxidase complex (ABC, Vector Laboratories; Burlingame, CA) and 3,3’-diaminobenzidine (DAB; Sigma, St. Louis, MO) with saturated nickel ammonium sulfate and imidazole. All solutions subsequent to proteinase K, and all wash steps, excluding DAB incubation, were carried out in 0.1 M PBS with 0.3% Triton X-100, pH 7.4. Sections were then counterstained in 1% Neutral Red. Positive neuronal perikarya and nerve fibers are bluish-black while background and negative tissue structures are red. Cases were staged with the Unified Staging System for Lewy Body Disorders (USSLB) [3,4].