The primary antibodies for αSYN (1:200; EP1536Y; AB-CAM, Cambridge, UK) and S-100 protein (1:200; 18-0046; Zymed, South San Francisco, CA, USA), with BenchMark autostainers (Ventana Medical Systems, Tuscan, AZ, USA), were used according to the manufacturers’ protocol. Then, 4-μm tissue sections were deparaffinized, and hydrated in xylene and serially diluted ethanol, respectively. Endogenous peroxidase was blocked by incubation in 3% H2O2 for 10 minutes, after which heat-induced antigen retrieval was performed for 32 minutes. After primary antibody incubation for 32 minutes at room temperature, detection was carried out with an I-View detection kit (BenchMark XT, Ventana Medical Systems).
The specimens were determined to be adequate for αSYN immunostaining when sufficient numbers of sections were available for analysis, and the density of S-100-positive nerve cells was at least low to moderate, with the presence of the muscularis mucosa. A section with at least one definite αSYN immunoreactive fiber was considered to be positive when compared with the positive control of αSYN immunoreactivity (ie, Lewy body-like inclusions or Lewy neurites from the brain tissues of PD patients;Fig. A ). The neuropathological findings were assessed by one neuropathologist (S.M.H.) and one experienced neurologist (J.K.) who were blinded to the clinical information of subjects.
The specimens were determined to be adequate for αSYN immunostaining when sufficient numbers of sections were available for analysis, and the density of S-100-positive nerve cells was at least low to moderate, with the presence of the muscularis mucosa. A section with at least one definite αSYN immunoreactive fiber was considered to be positive when compared with the positive control of αSYN immunoreactivity (ie, Lewy body-like inclusions or Lewy neurites from the brain tissues of PD patients;