Immunohistochemical Analysis of Neurodegenerative Markers

Immunohistochemistry against α-syn, phosphorylated α-syn (p-α-syn), tau and phosphorylated tau (p-tau) was performed on sections of appendix and cecum as previously described [21 (link), 22 (link)]. Sections were deparaffinized and treated for heat antigen retrieval in a microwave for 6 minutes at 100% power followed by 3 minutes at 60% power and left to cool for 1 hour at room temperature. The sections were then washed and endogenous peroxidase activity blocked by incubation in a mix of 30% H₂O₂ and 60% methanol. Nonspecific binding sites were blocked with Super Block (ScyTek, Logan, UT) for 60 minutes at room temperature and then incubated overnight with primary antibody (Table 2). The primary antibody was diluted in blocking buffer plus 0.1% Triton-X. The sections were then incubated in an appropriate biotinylated secondary antibody (1:200), followed by avidin-biotin-complex peroxidase (VECTASTAIN Elite ABC HRP Kit, Vector Laboratories, Burlingame, CA) and visualized with a commercial 3,3’-diaminobenzidine (DAB) kit (Vector Laboratories, Burlingame, CA). Appendix and cecum sections were counterstained with Hematoxylin, dehydrated and coverslipped (Cytoseal mounting medium, Thermo Scientific, Waltham, MA, USA).