Mouse anti tau

Brain tissues were fixed in 4% paraformaldehyde and embedded in paraffin blocks. After deparaffinization and rehydration, brain slices were boiled in citric buffer (10 mM Citric acid, pH 6.0) for 4 min for antigen retrieval. The slides were incubated with 3% hydrogen peroxide for 30 min to block endogenous peroxidase activity. Subsequently, sections were incubated overnight at 4°C with the following primary antibodies: rabbit anti-β-Amyloid (1:400; Cell Signal Technology), mouse anti-Tau (1:1,000; Cell Signal Technology) and rabbit anti-P-Tau Thr181 (1:1,000; Cell Signal Technology). Following removal of the antibodies via several rinses with PBS, the respective antibodies were detected using the avidin-biotin-peroxidase complex method. Slides were lightly counterstained with hematoxylin and then were dehydrated with sequential ethanol. All images were analyzed by optical microscopy.

Labelling for Syp, Map2 and Tau proteins was performed on free-floating brain tissues. The tissues were treated with 0.1% sodium citrate at 4 °C for 30 min, incubated in 0.1% Triton X-100 in PBS for 30 min at room temperature and blocked with blocking solution (5% bovine serum albumin, 2% normal goat serum, 0.1% Triton X-100 and 0.05% Tween 20 in PBS) for 1 h before incubation with the primary antibodies. The tissues were incubated overnight with monoclonal mouse anti-Syp (1:500; Santa Cruz Biotechnology SC-123737), mouse anti-Map2 (1:500; Santa Cruz Biotechnology SC-51669) and mouse anti-Tau (1:1000; Cell Signaling 4019) antibodies in 50% blocking solution with PBS. The tissues were rinsed three times in PBS for 10 min each and incubated in the dark with Alexa 647-conjugated anti-mouse IgG (1:500, Thermo Fisher Scientific) secondary antibodies for 1 h. After washing, the nuclei were stained for 5 min in a 1 µL/mL 6-diamidino-2-phenylindole-dihydrochloride (DAPI) solution (Boehringer Mannheim GmbH, Mannheim, Germany), and the tissues were coverslipped with fluorescence mounting medium (DAKO, Santa Clara, CA, USA). The control tissues were treated in the same way but without the primary antibodies. Images were collected with a Nikon confocal microscope.