Anti p tau

At the end of the final behavioral test, mice were anesthetized and transcardially perfused with NS and 4% paraformaldehyde in phosphate-buffered saline (PBS). Brains were sliced (10 µm) and analyzed by immunohistochemistry. Briefly, slides were blocked with 1% BSA (Sigma) for 1 h, and permeated with 0.3% Triton X-100 in 1% BSA/PBS for 30 min at RT. Sections were incubated with anti-Aβ_1–42 (1:1,000; EMD Millipore, Billerica, MA, USA), anti-p-Tau (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-BACE (1:1,00; Cell Signaling Technology, Inc.), anti-synaptophin (1:1,000), anti-AMPAR-1 (1:1,000), anti-AMPAR-2 (1:1,000; all from Abcam, Cambridge, MA, USA), anti-BDNF (1:1,000; Promega Corporation, Madison, WI, USA), and anti-GDNF (1:1,000; Promega Corporation) at 4°C overnight. After washing, the slices were incubated with secondary antibodies at RT for 2 h. The nucleus was stained by Hoechst 33342 (1 µg/ml, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Control sections were run following identical protocols, but the primary antibodies were omitted. Results were visualized under fluorescence microscopy by IMAGE-PRO PLUS software (Media Cybernetics, Inc., Rockville, MD, USA). Quantification analysis of positive cells was performed on three sections per animal, and four mice per group were analyzed in a blinded fashion.