Brain and spinal cord tissue were fixed in 10 % formalin and embedded in paraffin. Sections (10 μm thick) were cut from the cerebrum, midbrain, pons, medulla oblongata, cerebellum, and spinal cord. The sections were stained with hematoxylin and eosin as well as Klüver–Barrera stain. Immunohistochemistry was performed for tau (AT8, 1:1,000; Thermo Scientific), α-synuclein (pSyn#64, 1:1,000; Wako), TDP-43 (409/410, 1:1,000; original antibody [22 (link)]), FUS protein (HPA008784, 1:1,000; Sigma-Aldrich; and A300-302A, 1:500–1,000; Bethyl Laboratories), Ewing sarcoma protein (EWS, 1:100; Santa Cruz Biotechnology), and TATA-binding protein-associated factor 15 (TAF15, 1:50; Bethyl Laboratories). Primary antibody labelling was visualized using 0.2 % 3,3′-diaminobenzidine as the chromogen in combination with an Envision Plus kit (Dako Japan, Tokyo), according to the manufacturer’s instructions.
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