Early Parkinson’s disease is defined by α-synuclein oligomer-driven aggregation susceptible cells

Formalin-fixed paraffin-embedded (FFPE) tissue sections were obtained from the cingulate cortex (see tables S2 and S3) and cut to 8 μm thickness. FFPE sections were baked at 37 ℃ for 24 hours followed by 60 ℃ overnight. Sections were deparaffinized in xylene, and rehydrated using graded alcohols. Non-specific binding was blocked with 10% bovine serum albumin (BSA) solution in PBS for 30 minutes. The tissue was then pressure cooked in citrate buffer at pH 6 for 10 minutes. Tissue sections were incubated with primary antibodies; anti-phosphorylated alpha-synuclein (ab59264, AB_2270761, Abcam, 1:200); and either ionized calcium-binding adapter molecule 1 (GT10312, Thermo Fisher, AB_2735228, 1:500) or Microtubule-Associated Protein 2 (ab254143, Abcam, AB_2936822, 1:500) for 1h at room temperature. The sections were then washed three times for five minutes in PBS followed by the corresponding AlexaFluor secondary antibodies (anti-rabbit 568, A11011, AB_143157, Thermo Fisher, anti-mouse 488, A11001, AB_2534069, Thermo Fisher, all at 1:200) for an additional hour at room temperature in the dark. Sections were then washed three times for five minutes again in PBS and incubated in Sudan Black (0.1% for 10 minutes, 199664-25G, Sigma Aldrich). Removal of Sudan Black occurred with three washes in 30% ethanol (E7148-500ML, Sigma Aldrich) before mounting with Vectashield+ (Vector Labs, H-1900) and coverslipping (VWR, 50 mm ✕ 24 mm #1 thickness, Catalogue Number 48404-453) for imaging. Sections were stored at 4 ℃ until imaging was completed.

Breiter JC, Beckwith JS, Brock EE, Lachica J, Toomey CE, Fu B, Weiss LE, Wood NW, Vendruscolo M, Gandhi S, Lee SF

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