Immunohistochemistry of FTLD-TDP superior frontal cortex was carried out as previously described (Phan et al., 2021 (link)). Briefly, formalin-fixed, paraffin-embedded sections (10 μm) were deparaffinized in xylene and rehydrated through graded ethanol, followed by antigen retrieval with citrate buffer (pH 6.0) using a pressure cooker (Aptum Bio Retriever 2,100, Aptum Biologics Ltd., United Kingdom) at a peak temperature of ~121°C and gradually cooling to room temperature. Endogenous peroxidase was blocked with 1% hydrogen peroxide in 50% ethanol. Sections were probed with TDP-43 antibody (Proteintech, 10,782-2-AP, 1:400) and NeuN antibody (Biolegend, SIG-39860, 1:100), washed with PBS and incubated with the corresponding secondary antibodies (Thermo Fisher Scientific, A-10042 and A-31571, 1:250) and 4′,6-diamidino-2-phenylindole DAPI (Sigma-Aldrich, D9542, 1 mg/ml). The slides were treated with 70% Sudan Black for 30 min and 10 mM CuSO4 in 50 mM ammonium acetate buffer (pH 5.0) to quench auto-fluorescence signals prior to cover-slipping with anti-fade fluorescence mounting medium (DAKO, S3023) and then sealed with nail polish. Negative controls (without primary antibodies or secondary antibodies) were performed for each immunohistochemistry run, and no signals were detected in each case.
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