Anti 6e10

The hippocampi were homogenized in a pro-prep solution (17081, iNtRon, Gyeonggi-do, Korea) after physically mincing. The protein concentration was quantified using the Bradford reagent (Bio-Rad, Hercules, CA, USA), and then denatured by boiling for 5 min, depending on the concentration of the sample. Each sample was separated by electrophoresis on 10–15% sodium dodecyl sulfate-polyacrylamide gels. Then, the proteins were transferred to a nitrocellulose membrane and blocked for 30 min with 5% bovine serum albumin (BSA) solution for nonspecific blocking. Membranes were incubated at 4 °C with anti-6E10 (1:1000, Sig-39320, Covance), anti-ADAM10 (1:1000, ab1997, Abcam, Cambridge, UK), anti-BACE (1:1000, sc-33711, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GFAP (1:1000, ab53554, Abcam), anti-Iba1 (1:1000, ab15690, Abcam), or anti-β-actin (1:3000, #3700, Cell Signaling, Danvers, MA, USA). After overnight incubation, membranes were washed thoroughly in PBS-T buffer and incubated for 1 h with the corresponding secondary antibody diluted at 1:3000. The membranes were then washed, and immunoreactivity was detected using an enhanced chemiluminescence (ECL) kit (NEL105001EA, Perkin Elmer, Waltham, MA, USA).

Monomeric and oligomeric Aβ₄₂ were prepared as described previously [36 (link)], from aliquots of the same batch of Aβ₄₂. For oligomeric Aβ₄₂, lyophilized Aβ₄₂ aliquots (0.3 mg) were dissolved in 0.2 ml of 1,1,1,3,3,3-Hexafluoro-2-propanol (HFP, Sigma–Aldrich) and then added to 0.7 ml H₂O. Samples were loosely capped and stirred on a magnetic stirrer under a fume hood for 48 h, and used within 36 h. Monomeric Aβ₄₂ was prepared immediately before use by rapidly evaporating the HFP via gentle bubbling of nitrogen gas into the solution. The quality of Aβ₄₂ preparations was checked by immunoblot with anti-A-11 (1:1000, Invitrogen) and anti-6E10 (1:1000, Covance, Princeton, NJ) antibodies.