Supersignal west femto sensitivity substrate

To characterize the proportions of Aβ monomers and oligomers in the solutions applied on cells, chronic and acute solutions were prepared by diluting Aβ_1–42 in RPMI medium (50 nM) or in PBS (1 μM). Samples were diluted in a reducing sample buffer (TrisHCl 63 mM, glycerol 30% , SDS 2% , Bromophenol blue 0.00025% , pH 6.8) and electrophoretically separated by SDS-PAGE using 4–20% Tris-Glycine polyacrylamide gels and Novex Tris Glycin running buffer (Life Technologies) at 125 mV for 1 h 40 min. The gels were transferred to nitrocellulose membranes (1 h 15 min at 125 mA) in transfer buffer (Tris 25 mM, Glycine 192 mM, SDS 0.02% , ethanol 20% ). The membranes were blocked in 0.5% nonfat dry milk in PBS-Tween20 0.1% and incubated for 15 min with an monoclonal anti-Aβ 6E10 primary antibody (Covance Inc.) and an HRP-conjugated Goat anti-mouse IgG secondary antibody (Jackson) using the SNAPi.d system (Millipore, Molsheim, France). Final detection was performed with chemioluminescence (Supersignal west femto sensitivity substrate, Thermo Scientific) using CL-Xposure films (Thermo Scientific).