Nitrocellulose membrane filters

Samples (conditioned media, CM, or cell lysates) were diluted in NuPAGE™ LDS sample buffer (ThermoFisher Scientific, NP0007) and 10% DTT solution 1 M (Applichem, Germany, A3668, 0050), boiled 10 min, loaded in precasted NuPAGE™ Novex™ 10% Bis-Tris Midi Protein Gels (ThermoFisher Scientific, WG1201A), running in NuPAGE MES SDS running buffer (ThermoFisher Scientific, NP0002). The semidry blot was then done onto nitrocellulose membrane filters, 0.22 μm (GE Healthcare, 10600001). The membrane filter was boiled in PBS to increase the detection of low molecular weight (MW) bands. After the incubation with the blocking solution (TBS 0.05% tween, 5% dry milk), primary antibodies were used in TBS 0.05% tween, 2.5% dry milk at the concentration reported in Table I. Following the incubation with secondary peroxidase-coupled anti-mouse or anti-rabbit antibodies, ECL (GE Healthcare, RPN2209) chemiluminescent detection was performed. Quantitative densitometric analysis was performed using ImageJ software (http://imagej.nih.gov/ij/), according to the procedure described in the dedicated section.

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.