Western blot analysis

At 6 w and 7 m after CCI, mice were euthanized with cervical dislocation without anesthesia or perfusion so as not to affect the authentic phosphorylation state of tau; tau is known to be hyperphosphorylated by anesthesia^89,90 and very rapidly dephosphorylated during post-mortem delay⁹¹. Mouse brains were promptly dissected and submerged in ice-cold phosphate-buffered saline (PBS), in which bilateral hippocampi were dissociated from other brain regions. The hippocampal tissue was flash-frozen in dry ice and stored in −80 °C until used for Western blots.

The hippocampus was selected for biochemical analysis because it is a well-known brain region actively involved in learning and memory and it exhibits protein expression profiles that may predispose it to early development of tau pathology in AD⁹². Hippocampal tissue was homogenized in 9 volumes of buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM β-mercaptoethanol, 50 mM NaF, 1 mM Na₃VO₄, 2.0 mM EDTA, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), and 10 μg/ml of each of aprotinin, leupeptin and pepstatin. The brain homogenates were mixed with 2-fold concentrated Laemmli buffer and boiled for 5 min, and the protein concentration was measured by using modified Lowry assay. The same amounts of protein from each sample were separated by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and electro-blotted onto PVDF membrane. After blocked with 5% fat-free milk, the membrane was incubated with primary antibodies (Table 1) overnight at room temperature in the presence of 0.1% NaN₃. After washed with three changes of TBST (Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20), the membrane was incubated with the corresponding HRP-conjugated secondary antibody for ~2 h at room temperature. After washed with TBST, the blots were visualized by enhanced chemiluminescence (Thermo Scientific, Rockford, IL) and quantified by densitometry using the Multi Gauge V3.0 software (Fuji Film Co., Ltd., Minato, Tokyo, Japan).

Primary antibodies employed in the present study.

Poly-, polyclonal; Mono-, monoclonal; S, sheep; R, rabbit; M, mouse; P-tau, phosphorylated tau.