Bace1 61 3e7

Total protein was extracted from 15 mg of whole-brain or hippocampus samples with protein 1× lysis buffer (2× lysis buffer, 100 mM of Na₃VO₄, and 0.5 M of NaF) with 1× protease inhibitor (Roche, switzerland). A total of 20 μg of protein samples was loaded on 10% or 15% SDS-PAGE gels (Bio-Rad). The loaded samples were then transferred to a PVDF membrane (Millipore, Billerica, MA, USA) in transfer buffer (Tris, pH 8.3, glycine, and 10% methanol) at 4 °C for 2 h. The transferred membrane was blocked with 5% skim milk (Merck) for 30 min at room temperature and incubated with specific primary antibodies overnight at 4 °C in TSBS with 1% skim milk. The primary antibodies of the following proteins were used for Western blotting: APP (NAB228, 1:500; Santa Cruz), ADAM10 (T-17, 1:500; Santa Cruz), BACE1 (61-3E7, 1:1000; Abcam), PSEN1 (N-19, 1:500; Santa Cruz), VDR (D-6, 1:500; Santa Cruz), RXRα (D-20, 1:1000; Santa Cruz), RXRβ (L-20, 1:1000; Santa Cruz), and β-actin (AC-15, 1:1000; Santa Cruz). After secondary antibody incubation for 30 min at room temperature, the membrane was developed with an LAS 4000 imaging system (Fujifilm, Japan). The quantification of the protein expression was calculated with the software Fiji [33 (link)].

Formalin-fixed brain tissues from the rats after feeding them a high-phytate diet for 18 weeks were dehydrated with ethanol, embedded in paraffin, and sectioned at 3 µm. Antigen retrieval was performed in Tris-EDTA, at pH 9.0 for 3 min. For Aβ staining, the brain sections were further incubated in 70% formic acid for 15 min [11 (link)]. IHC was performed with antibodies on the following proteins at 25 °C for 2 h: Aβ (4G8, 1:200; Covance, US), BACE1 (61-3E7, 1:200; Abcam), PSEN1 (N-19, 1:200; Santa Cruz, Dallas, TX, USA), and cleaved caspase 3 (D175, 1: 200 Cell Signaling Technology, MA, USA). Horseradish peroxidase-conjugated secondary antibodies were detected with 3,3,-diamino-benzidine (DAB) tetrahydrochloride substrate (Dako, Glostrup, Denmark) or alkaline phosphatase (AP, GBI Labs, WA, USA). All sections were counterstained with hematoxylin (Thermo Scientific, MA, USA). Images were captured with a 3DHISTECH microscope (Budapest, Hungary).