To examine the tau pathology in brains, fixed hemi-brains were embedded in paraffin blocks and sectioned coronally at 4 μm thickness for histochemical analysis. De-paraffinized brain sections were stained with H&E or immunostained using AT8 antibody (MN1020B; Thermo Fisher Scientific), PHF-1 antibody, MC1 antibody (kind gifts from Dr. Peter Davies, Albert Einstein College of Medicine, Bronx, NY), LRP1 antibody (ab92554; Abcam), NeuN antibody (MAB377; Sigma-Aldrich), or AQP4 antibody (A5971; Sigma-Aldrich). For antigen retrieval, de-paraffinzed sections were treated with microwave (550 W, 10 min) in citrate buffer (pH 6.0) prior to immunostaining. To detect proteinase K resistant tau inclusion, sections were treated with 100 µg/ml proteinase K for 6 min at 37°C prior to AT8 and PHF-1 staining (Iba et al., 2013 (link)).
To detect tau accumulation, isolated dcLNs were embedded in 4% NuSieve GTG agarose after carefully removing fatty tissue and sliced at 100 μm thickness with a LinearSlicer PRO7 (Dosaka EM). The sections were blocked with 10% calf serum for 30 min and incubated with anti–LYVE-1 antibody (ab14917; Abcam) and washed and incubated with goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (A-21245; Thermo Fisher Scientific). The sections were then mounted with PROLONG Anti-Fade Gold with DAPI (Thermo Fisher Scientific).
To detect tau accumulation, isolated dcLNs were embedded in 4% NuSieve GTG agarose after carefully removing fatty tissue and sliced at 100 μm thickness with a LinearSlicer PRO7 (Dosaka EM). The sections were blocked with 10% calf serum for 30 min and incubated with anti–LYVE-1 antibody (ab14917; Abcam) and washed and incubated with goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 (A-21245; Thermo Fisher Scientific). The sections were then mounted with PROLONG Anti-Fade Gold with DAPI (Thermo Fisher Scientific).
