A10042

Coronal brain slices of 40 μm were obtained using a freezing microtome (Leica) and collected in 0.1 M phosphate-buffered saline (PBS) solution. Brain slices were washed in PBST (1% Triton in 0.1 M PBS) for 40 min. A blocking solution (10% donkey serum, 0.2% Triton in 0.1 M PBS) was applied for 2 h at room temperature. Brain sections were incubated with primary antibodies (chicken anti-GFP, 1:200, Abcam, ab13970; rabbit anti-tyrosine-hydroxylase, 1:200, Millipore, AB152) overnight at 4°C followed by secondary antibodies for 2 h at room temperature. The secondary antibodies used were donkey anti-rabbit conjugated with Alexa Fluor 568 (1:200, Invitrogen, A10042) and donkey anti-chicken CF488 (1:200, Sigma-Aldrich, SAB4600031). Brain slices for DAPI staining were incubated with DAPI (1:1000, Sigma) for 20 min. Finally, these slices were mounted on coverslips and imaged. Images were obtained by the use of a scanning confocal microscope (TCS SP5, Leica) and LAS AF software (Leica).

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.