Immunostaining of Phospho-Tau and Antioxidant Proteins

Deparaffinization and antigen retrieval of the brain tissue sections were performed as previously described.^{30 (link)} After washing with phosphate-buffered saline (PBS), sections were blocked in 0.1% Triton X-100 (Sigma-Aldrich), 2% normal goat serum (Thermo Fisher Scientific, MA, USA) and 1% bovine serum albumin (Sigma-Aldrich) in PBS for 1 h at room temperature. Sections were then incubated with primary antibodies diluted in blocking buffer overnight at +4°C. After 3 × 10 min washes in 0.1% Triton X-100 in PBS, they were incubated with secondary antibodies and DAPI (2-(4-Amidinophenyl)-1H-indole-6-carboxamidine) for 1 h at room temperature followed by 3 × 10 min washes in Triton X-100 in PBS. The following dilutions and antibodies were used: 1:100 mouse anti-phospho-tau (Thr212, Ser214) (MN1060; Thermo Fisher Scientific), 1:100 rabbit anti-TRX-1 (MA532569; Thermo Fisher Scientific), 1:100 rabbit anti-AGT (MA529010; Thermo Fisher Scientific), 1:500 Alexa Fluor fluorescently conjugated secondary goat anti-rabbit and anti-mouse antibodies (Thermo Fisher Scientific) and 1:100 DAPI (1351303; Bio-Rad, USA). To quench the autofluorescence of lipofuscin, sections were washed for 5 min in PBS and incubated with 0.1% w/w Sudan Black B (199664; Sigma-Aldrich) in 70% ethanol. Prior to mounting, sections were washed with Triton X-100 in PBS 2 × 10 min and 10 min with PBS.