2.5. Western blot for phosphorylated Tau

Frozen hippocampal tissues were homogenized as described previously [43]. Total amounts of 40 g protein per sample were separated on 10% sodium dodecylsulfate-polyacrylamide gels, and transferred to nitrocellulose membranes (BioRad Laboratories). After blocking, membranes were incubated overnight at 4 ◦C with primary antibodies to phosphorylated Tau (p-Tau) S202/T205 (clone AT-8; 1:500, Fisher Scientific) and p-Tau pT231 (clone AT180; 1:1000, Innogenetics). After a washing step, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (GE Healthcare) for 2 h at room temperature. Blots were developed with electrochemiluminescence (ECL Prime, GE Healthcare) and visualized using a CCD camera (LAS-3000, Fuji Film). Density of bands was analyzed with Multi Gauge (version 3.0) software (Fuji Film). All samples were run in one blot for each marker. -actin was used as a loading standard, and protein levels were normalized to -actin levels in the respective blots.