CM and cellular samples were spotted onto nitrocellulose membrane filters 0.22 μm (GE Healthcare, 10600001). After incubation with the blocking solution (TBS 0.05% tween, 10% dry milk or TBS 0.01% tween, 10% dry milk for anti-oligomer pAbA11) primary antibodies were used in TBS 0.05% tween, 5% dry milk or TBS 0.01% tween, 5% dry milk (for anti-oligomer pAbA11). Purified recombinant anti-AβOs scFvA13 (3.5 μg/ml) was used as described [24 (link)] and immunodetection was performed by anti-V5 tag (Fig. 2e ) or by anti-His tag (Fig. 4c ), which respectively recognizes the V5 tag or C-terminal 6xHis tag of recombinant scFvA13. After incubation with secondary peroxidase coupled anti-mouse or anti-rabbit antibodies, ECL (GE Healthcare, RPN2209) chemiluminescent detection was performed. Serial dilution curves of cellular samples were preliminarily tested to obtain nonsaturating condition of immunodetection and samples were loaded at 1 μg/spot, whereas 1 μl of CM were spotted. For each specific antibody staining protein loading was normalized to the corresponding Ponceau staining. Blots were scanned and quantitative densitometric analysis was performed by using ImageJ software (http://imagej.nih.gov/ij/ ), as described in Supplementary Materials and Methods.
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