Anti aβ antibody 82e1

A solution of 7 (1 mM in 10% DMSO/15% Kolliphor EL/75% PBS, 0.2 ml, ~4.0 mg/kg) was intravenously injected into 11-month-old App knock-in (App^{NL-G-F/NL-G-F}) mice expressing human Arctic Aβ. After 60 min, the mice were irradiated with LED (λ = 600 nm) for 10 min. The operation set (catalyst injection and photoirradiation) was repeated five times over 5 days. At 24 hours after the final operation set, the hippocampus was excised. The hippocampus was homogenized using a 10× volume of tris buffered saline (TS) buffer [50 mM tris, 150 mM NaCl (pH 7.6), cOmplete EDTA+ (Roche)]. After the mixture was centrifuged (260,000g, 20 min, 4°C), the supernatant was removed, and the precipitate was homogenized using 2% Triton-X–containing TS buffer (equal volume as the TS buffer above). The mixture was then centrifuged (260,000g, 20 min, 4°C), and again, the supernatant was removed. The resulting precipitate was homogenized using 70% FA, sonicated, and centrifuged (260,000g, 20 min, 4°C). The collected supernatant (=FA fraction) was evaporated, redissolved in DMSO, and analyzed by Western blotting using anti-Aβ antibody (82E1; IBL). The protein subjected to SDS-PAGE was quantified using the bicinchoninic acid method, and the applied amount was normalized.

The brain excised from a 7-month-old App knock-in (App^{NL-G-F/NL-G-F}) mouse expressing human Arctic Aβs (29 (link)) was separated into cortex, hippocampus, and remaining brain tissue. The cortex and hippocampus were combined and homogenized in PBS (8 mM Na₂HPO₄·12H₂O, 2 mM NaH₂PO₄·2H₂O, 130 mM NaCl), and the suspensions were stored at −80°C until use. 7 was added to the PBS-lysate suspension (final 50 μM) and photoirradiated with LED (λ = 595 nm) or kept in the dark at 37°C. At certain time points, an aliquot of the reaction mixture was diluted with FA (final 70% concentration), evaporated, and redissolved in DMSO. The solution was analyzed on 15% acrylamide/bis mixed solution (29:1) (Nacalai Tesque), with 0.1% SDS running buffer under reducing (1% 2-mercaptoethanol) condition. Molecular weight was estimated with Precision Plus Protein Standards dual color (Bio-Rad, California, USA). Western blotting analysis was performed using anti-Aβ antibody (82E1; IBL).