All brain tissues were assessed by immunohistochemistry, as previously described in Gary et al. 2019 [11 (link)]. Briefly, 4-μm-thick paraffin sections were cut, deparaffinized in xylene, successively rehydrated in ethanol (100, 90, and 70%) and rinsed under running tap water for 10 min before immunohistological staining. They were then incubated in 99% formic acid for 5 min, quenched for endogenous peroxidase with 3% hydrogen peroxide and 20% methanol, and washed in water. Sections were blocked at room temperature for 30 min in 4% bovine serum albumin (BSA) in 0.05 M tris-buffered saline, with 0.05% Tween 20, pH 8 (TBS-Tween, Sigma). They were then incubated overnight at + 4 °C with the 6F3D anti-Aβ antibody (Dako, 1/200), polyclonal anti-tau antibody (Dako, 1/500), monoclonal anti-alpha-synuclein (LB509, Zymed, 1/250), polyclonal anti-TDP43 (Protein Tech Group, 1/1000) routinely used for Aβ, tau, alpha-synuclein and TDP43 detection, respectively. Sections were further incubated with a biotinylated secondary antibody for 25 min at room temperature, and the presence of the secondary antibody was revealed by a streptavidin–horseradish peroxidase conjugate using diaminobenzidine (Dako, Glostrup, Denmark). Sliced were counterstained with Harris hematoxylin.
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