Immunohistochemistry

Immunohistochemistry was performed as described previously (Yin et al., 2017). Briefly, 16 μm sections of PFA-fixed human brain tissue were vacuum-dried, post-fixated for 10 min with 4% PFA, and washed with PBS. Heat-induced antigen retrieval was performed in sodium citrate solution (pH 6.0) for 10 min in a microwave. Endogenous peroxidase was blocked by incubating the slides in 0.3% H₂O₂ for 30 min. After three washing steps with PBS, primary antibodies against IBA1 (WAKO, 019-19741, 1:1,000), Phospho-TAU (Thermo Fisher Scientific, Waltham, MA, USA, MN1020, clone AT8, 1:750), and Beta-Amyloid (Dako, M0872, 1:100) were diluted in Bright Diluent (ImmunoLogic, BD09-500) to prevent background staining and incubated overnight at 4°C. After three washing steps in PBS, secondary biotinylated horse antimouse IgG antibody (0.000125 mg/ml Vector BA-2001) was incubated for 1 h at room temperature. The tissue sections were washed three times in PBS. The signal was amplified with avidin-biotin complexes (Vectastain Elite ABC-HRP, Vector, PK-6100) for 30 min at RT and visualized with 3,3′-diaminobenzidine (Sigma–Adrich, St. Louis, MO, USA, D-5637). Additionally, after the phospho-TAU staining, we performed a crystal violet counterstaining. The slides were dehydrated with an ethanol series (50%, 70%, 80%, 90%, 2× 96%, and 3× 100% ethanol) and air-dried for 30 min prior to mounting a coverslip with DePeX (Serva, 18243). Imaging was performed with a Hamamatsu Nanozoomer at 40x magnification.