Horseradish peroxidase conjugated goat anti mouse secondary antibody

The Aβ samples from in vivo or in vitro experiments were analysed by gel electrophoresis followed by western blotting using an anti-Aβ antibody (6E10)24 (link)25 27 (link)28 (link)29 (link). Samples (10 μl) were separated on a 10–20% Tris-tricine gel (Invitrogen, Grand Island, NY, USA). Following separation, the proteins were transferred onto nitrocellulose membranes and blocked with bovine serum albumin (BSA, 3% w/v, Sigma-Aldrich, St Louis, MO, USA) in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) for 2 h at room temperature or overnight at 4 °C. The membranes were incubated with an anti-Aβ antibody (6E10, 1:2,000, Covance, Princeton, NJ, USA) in a solution of 2% BSA (w/v in TBS-T) for 4 h at room temperature or overnight at 4 °C. After washing with TBS-T (3 × , 10 min), a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1:5,000 in 2% BSA w/v in TBS-T; Cayman Chemical Company, Ann Arbor, MI, USA) was added for 1 h at room temperature. The Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA), Biosesang ECL Plus kit (Biosesang, Gyeonggi-do, Republic of Korea), or a homemade ECL kit53 (link) was used to visualize the results on a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).

The samples from the inhibition and disaggregation experiments were analyzed by gel electrophoresis with Western blot using an anti-Aβ antibody (6E10)20 (link)28 (link). Each sample (10 μL) was separated on a 10–20% Tris-tricine gel (Invitrogen, Grand Island, NY, USA). Following separation, the gel was transferred onto nitrocellulose membrane which was blocked with bovine serum albumin (BSA, 3% w/v, Sigma-Aldrich, St. Louis, MO, USA) in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) for 3 h at room temperature. The membrane was treated with antibody (6E10, Covance, Princeton, NJ, USA; 1:2000) in a solution of BSA (2% w/v) in TBS-T overnight at 4 °C. Following washing, the membrane was treated with horseradish peroxidase-conjugated goat antimouse secondary antibody (1:5000; Cayman Chemical, Ann Arbor, MI, USA) in 2% BSA in TBS-T solution for 1 h at room temperature. Protein bands were visualized using ThermoScientific Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA).