Supplemental E-Methods

Supplemental e-methods

PET blot

6 µm serial tissue sections were collected using a microtome throughout the full extent of each biopsy specimen. Tissue was deparafinized and dried for 2 days at 60°C prior to PET blotting.Tissue sectionswere layered on nitrocellulose membranes, pre-wetted in Tris buffered saline (TBS) and digestedwith 250µg/ml proteinase-K in digestion buffer (10 mMTris-HCl, pH7.8, 100 mMNaCl, and 0.1% Brij 35) for 8 h at 55°C. After washing, themembranes were treated for 10 min with 3M guanidine isothiocyanate for epitope retrieval. Immunodetectionof α-syn aggregates was performed using two independent sources of LB509 (Abcam, ab27766, 1:100 and LB509, Zymed Laboratories, both 1:10,000 for 1 h) and alkaline phosphatase-coupled rabbit anti-mouse antibody as a secondaryantibody. Both primary antibodies showed identical results. Immunodetectionof ser-129p-α-syn aggregates was performed using and antibody from Abcam (ab59264, 1:100). Aggregates were visualized bythe formazan reaction using nitro-blue tetrazolium chloride (Roche, Mannheim, Germany) and 5-bromo-4-chloro-3-indoyl-phosphate p-toluidine salt(Roche) in NTM buffer (10 mMTris-HCl, pH9.5, 100 mMNaCl, and 50 mM MgCl2). All washes were carried out in TBS.

Immunohistochemistry

Immunohistochemistry for α-syn (LB509; Abcam, ab27766, 1:100 and LB509, 1:2000; Zymed Laboratories), and ser-129p-α-syn (Abcam, ab59264, 1:100) was carried out in serial sections. Both primary antibodies for α-syn showed identical results. Briefly, sections were de-parffinized, rehydrated in a series of alcohols and washed. Endogenous peroxidase activity was blocked with 3% aqueous hydrogen peroxide. Antigen retrieval was carried out according to manufacturer’s recommendations; in a decloaking chamber (Biocare) using citrate buffer (pH 6.0) and Tris-EDTA buffer (pH 9.0) for LB509 or sections were digested in 1% pepsin in 0.01N HCl at 37 C for 15 mins for ser-129p-α-syn. All sections were blocked in 2.5% normal horse serum. Sections were incubated in primary antibodies overnight at room temperature. Sections were washed in 1xTBS. Staining was completed using Super Sensitive Polymer HRP kit (Biogenex: BGQD440-XAK) according to manufacturers’ instructions. Finally sections werewashed in 1x TBS and developed with freshly prepared DAB solution before dehydration in graded alcohols, clearing in xylene and mounting in Permount (Fisher). Appropriate controls to test specificity of α-syn antibodies consisted of the use of α-syn blocking peptides (Abcam, ab105629 and ab123758) and/or omission of primary antibodies. In some of the sections prepared for PET blot, we used non-aggregating proteins glial fibrillary acidic protein (GFAP; Abcam, ab7260; 1:5000) to ensure that immunopositive profiles were not non-specific. Brain tissue from pathologically proven PD cases and individuals without PD was used for PET blot optimization and as positive and negative control tissue respectively.

Immunohistochemistry and PET blot control studies I

Control and PD brain tissue was used to optimize the PET blot for detection of pathologic α-syn (Figure e-1). As is well established, conventional immunohistochemistry for α-syn detected weak levels of physiologic α-syn in brain tissue from healthy individuals without PD with no evidence of Lewy bodies or Lewyneurites and a strong background (Figure e-1A). In brain tissue from individuals with PD conventional immunohistochemistry for α-syn detects Lewy bodies (Figure e-1B), which can be identified by their distinct morphology at higher magnification (Figure e-1C). Following PET blot for α-syn there is a complete lack of signal for α-syn in control brain tissue (Figure e-1D), suggesting the PET blot has degraded physiologic α-syn in control cortex. Unlike when conventional immunohistochemistry is applied (Figure e-1A) there is no background staining apparent for α-syn when using the PET blot (Figure e-1D) evidencing the increased specificity of this approach. The superior antigen retrieval of the PET blot also increases the sensitivity for detecting aggregated proteinase-K resistant α-syn PD brain issue (Figure e-1E, F) with clear labeling of Lewy bodies and Lewyneuritesbeing more apparent compared to that seen with conventional immunohistochemistry in adjacent sections (Figure e-1 B and C respectively). Similar findings were also apparent for ser-129p-α-syn in brain (data not shown).

The PET blot specifically detects pathologic α-syn in PD midbrain. Control and PD midbrain tissue was used to optimize the specificity of the PET blot for detection of pathologic α-syn aggregates. The PET blot detected several regions of dense positivity for α-syn in the PD midbrain (Figure e-2A). These aggregates in some cases were distributed in close proximity to pigmented neurons (Figure e-2B). In contrast, in control midbrain using the PET blot there was no positivity for α-syn (Figure e-2C, D) This finding, in control midbrain, suggests that all non-pathologic α-syn had been destroyed by the PET blot method thus demonstrating the enhanced specificity of this method for detection of aggregated α-syn.

Specificity of the PET blot for detection of α-syn in colonic mucosal biopsy tissueTwo types of control study were conducted to verify the specificity of the PET blot for the detection of α-syn and ser-129p-α-syn in colon biopsy tissue. The first control made use of commercially available blocking peptides specific for the α-syn and ser-129p-α-syn antibodies employed in the present study. Figure e-3A illustrates staining for α-syn by conventional immunohistochemistry in a colon biopsy from a control subject. An area of strong positivity is shown in higher magnification in Figure e-3D. Staining for α-syn in an adjacent section using the PET blot shows a high degree of overlap (Figure e-3B), with the same region showing a strong signal for α-syn as shown at higher magnification (Figure e-3E). Pre-incubation of the primary antibody for α-syn with a blocking peptide eliminates all staining for α-syn by PET blot (Figure e-3C and at high magnification in Figure e-3F). Similar findings were evident for ser-129p-α-syn (data not shown). The second control consisted of carrying out the PET blot using our standard method but with the primary antibody for α-syn or ser-129p-α-syn omitted. Figure e-3G illustrates ser-129p-α-syn staining by PET blot with primary antibody included and Figure e-3H illustrates staining in an adjacent section with the primary antibody omitted. Omission of the primary antibody clearly eliminated all signal for ser-129p-α-syn by PET blot. Similar findings were evident for α-syn (data not shown). Taken together these controls demonstrate that using the currently applied methodology, the PET blot is a specific method for the detection of both α-syn and ser-129p-α-syn in colon biopsy tissue.