*2.5. Enzyme-Linked Immunosorbent Assay (ELISA) Quantitation of A*β

In order to measure the Aβ levels in the hippocampus, hippocampal tissue was isolated on ice and homogenized in a lysis buffer (T-PER® Tissue Protein Extraction Reagent; Thermo Fisher Scientific, Waltham, MA, USA) containing a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and a phosphatase inhibitor cocktail (Nacalai Tesque) with a pellet mixer. After incubation on ice for 15 min, the lysates were centrifuged at 14,200× *g* for 5 min at 4 ◦C and the supernatants were placed in a fresh tube. Protein concentration was determined using a Bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The levels of T-PER-extractable Aβ were measured with the Human β Amyloid (1–40) enzyme-linked immunosorbent assay (ELISA) Kit Wako II (#298-64601; Fujifilm Wako Pure Chemical Corp., Osaka, Japan), the Human β Amyloid (1–42) ELISA Kit Wako, High Sensitivity (#296-64401, Fujifilm Wako Pure Chemical Corp.), the Human/Rat/Mouse β Amyloid (1–40) ELISA Kit Wako II (#294-64701; Fujifilm Wako Pure Chemical Corp., Osaka, Japan), and the Human/Rat/Mouse β Amyloid (1–42) ELISA Kit Wako, High Sensitivity (#292-64501, Fujifilm Wako Pure Chemical Corp.) in accordance with the manufacturers' instructions.
