Anti tau phospho t181

Anesthetized mice were fixed by cardiac perfusion of phosphate-buffered saline (PBS) and 4% paraformaldehyde in PBS. Mouse brains were carefully dissected, postfixed for 24 h at 4°C in the same fixative solution, and incubated in 20% sucrose at 4°C until equilibration. The fixative was discarded by aspiration, and the brains were washed with PBS. Sequential 30 μm-thick coronal sections were obtained using a cryostat (CM1850UV; Leica Microsystems GmbH, Wetzlar, Germany) at 22°C. The sections were blocked in 5% normal goat serum and 5% normal horse serum (VECTOR Laboratories, Burlingame, CA, USA). The tissues were permeabilized with 0.3% Triton X-100 and immunofluorescence was performed using standard methods with the following antibodies: anti-human mitochondria (Merck Millipore, Burlington, Massachusetts, USA), anti-3R-tau (Wako), anti-Tau (phospho T181; Abcam), and anti-tau (phospho S404; Abcam). Alexa 488 and Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Europe Ltd., Newmarket, United Kingdom) were used to visualize the immune complexes. Antibody labeling was visualized under an LSM 800 confocal microscope (Carl Zeiss AG, Jena, Germany).

Cells and tissue lysates were prepared by ultrasonication (Branson Ultrasonics, Slough, United Kingdom) in buffer containing 9.8 M urea, 2.8 M thiourea, 4% 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate, 130 mM dithiothreitol (DTT), 40 mM Tris-Cl (pH 8.8), and 0.1% sodium dodecyl sulfate. Protein levels were measured using Bradford assays (Bio-Rad Laboratories, Inc., Hercules, CA). For the immunoblot analysis, BOLT 4%–12% Bis-Tris gels (Life Technologies, Carlsbad, CA, USA) were electrophoretically transferred to nitrocellulose membranes. Each membrane was blocked in 5% skimmed milk and incubated with primary antibodies overnight at 4°C. After reaction with human recombinant protein-conjugated secondary antibodies at room temperature (RT) for 1.5 h, the immunoreactivity was detected using an ECL detection kit (GE Healthcare Life Sciences, Little Chalfont, UK). The antibodies used were as follows: anti-Tau (phospho T181; Abcam, Cambridge, United Kingdom), anti-Tau (phospho T231; Abcam), anti-Tau (phospho S396; Abcam), anti-Tau (phospho S404; Abcam), anti-glycogen synthase kinase 3 beta (GSK-3β; phospho Y216; Abcam), anti-total GSK-3β (Abcam), and anti-total tau (Wako, Osaka, Japan).