T NTA 2.2 software was used for data analysis.OC serum dot blotThe anti-amyloid fibril OC rabbit serum (Millipore) [21] was used at 1:1000 dilution according to the manufacturer’s instructions. Samples were diluted to 5 mM monomer concentrations and 2.5 mL of each sample was loaded onto untreated cellulose nitrate Protran BA85 membranes (Schleicher Schuell, Germany) and allowed to dry. An HRP-conjugated goat anti-rabbit IgG antibody (H+L, Invitrogen) was used to detect bound OC antibodies using chromogenic 3,3′,5,5′-tetramethylbenzidine (NovexH, Invitrogen) as substrate.SynaptotoxicityThe effect of Ab42CC protofibrils on spontaneous Title Loaded From File synaptic activity was evaluated in an in vitro microelectrode array (MEA) assay [9]. Soluble oligomers of Ab42 were used for comparison. These were prepared as described previously (Ref. [9]; the 10:0 Ab42: Ab40 ratio oligomers), with the modification that a 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl was used to match the Ab42CC buffer. Primary hippocampal neurons were dissected from e17 FVB mouse embryos and plated on MEA substrate (Multichannel Systems GmbH, Germany) at a density of 1000 cells mm22 (500,000 cells per chip). The spontaneous firing of neuronal networks was recorded after 1 to 2 weeks in culture. A temperature controller (Multichannel Systems) was used to maintain the MEA platform temperature at 37uC during the experiments. First, the basal firing rate was recorded for 500 s, then 0.5 mM of either Ab42 oligomers or Ab42CC protofibrils was added to MEA dish and neuronal activity was recorded for the next 500 s. The same amounts of Ab was added two more times to reach final concentration of 1.5 mM. Signals from active electrodes were amplified by means of a MEA1060 amplifier (gain 1200) and digitized by the A/D MC_Card at a sampling rate of 25 kHz. The MC_Rack 3.5.10 software (Multichannel Systems) was used for data recording and processing. The raw data were high-pass filtered at 200 Hz, and the threshold for spike detection was set to 5 standard deviations from the average noise amplitude computed during the first 1000 ms of recording. Numbers of spikes detected by every active electrode per time bin of 500 s were normalized to baseline (firing rate in the absence of treatment). The firing rates Title Loaded From File corresponding to 500 s treatments with 0.5, 1 and 1.5 mM of protofibrils/oligomers were computed and presented as percentage of initial rates. Use of animals and procedures were approved by the Ethical Committee for Animal Welfare (ECD, Ethische Commissie Dierenwelzijn) of KULeuven and IMEC. Timely pregnant FVBAtomic force microscopyConcentrated protofibrils or fibrils of Ab42CC, Ab42, or Ab40 were diluted to 0.5 to 1 mM in 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl, and 5 mL solutions were loaded onto freshly cleaved mica. After 1 to 2 min, the mica surface was briefly washed with 100 mL deionized water and air-dried. The samples were imaged immediately in AC-mode using a Cypher AFM instrument (Asylum Research, USA) equipped with NSC36/ Si3N4/AlBs three-lever probes (mMasch). The probes had nominal spring constants of 0.6 to 1.8 N/m and driving frequencies of 75 to 155 kHz. To determine protofibril length distributions, a number of images covering 1 to 2 mm2 surfaces were scanned and the lengths of particles were measured using a freehand tool in the MFP-3DTM offline section analysis software. The same tool was used to measure cross sections of particles.Analytical ultrace.T NTA 2.2 software was used for data analysis.OC serum dot blotThe anti-amyloid fibril OC rabbit serum (Millipore) [21] was used at 1:1000 dilution according to the manufacturer’s instructions. Samples were diluted to 5 mM monomer concentrations and 2.5 mL of each sample was loaded onto untreated cellulose nitrate Protran BA85 membranes (Schleicher Schuell, Germany) and allowed to dry. An HRP-conjugated goat anti-rabbit IgG antibody (H+L, Invitrogen) was used to detect bound OC antibodies using chromogenic 3,3′,5,5′-tetramethylbenzidine (NovexH, Invitrogen) as substrate.SynaptotoxicityThe effect of Ab42CC protofibrils on spontaneous synaptic activity was evaluated in an in vitro microelectrode array (MEA) assay [9]. Soluble oligomers of Ab42 were used for comparison. These were prepared as described previously (Ref. [9]; the 10:0 Ab42: Ab40 ratio oligomers), with the modification that a 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl was used to match the Ab42CC buffer. Primary hippocampal neurons were dissected from e17 FVB mouse embryos and plated on MEA substrate (Multichannel Systems GmbH, Germany) at a density of 1000 cells mm22 (500,000 cells per chip). The spontaneous firing of neuronal networks was recorded after 1 to 2 weeks in culture. A temperature controller (Multichannel Systems) was used to maintain the MEA platform temperature at 37uC during the experiments. First, the basal firing rate was recorded for 500 s, then 0.5 mM of either Ab42 oligomers or Ab42CC protofibrils was added to MEA dish and neuronal activity was recorded for the next 500 s. The same amounts of Ab was added two more times to reach final concentration of 1.5 mM. Signals from active electrodes were amplified by means of a MEA1060 amplifier (gain 1200) and digitized by the A/D MC_Card at a sampling rate of 25 kHz. The MC_Rack 3.5.10 software (Multichannel Systems) was used for data recording and processing. The raw data were high-pass filtered at 200 Hz, and the threshold for spike detection was set to 5 standard deviations from the average noise amplitude computed during the first 1000 ms of recording. Numbers of spikes detected by every active electrode per time bin of 500 s were normalized to baseline (firing rate in the absence of treatment). The firing rates corresponding to 500 s treatments with 0.5, 1 and 1.5 mM of protofibrils/oligomers were computed and presented as percentage of initial rates. Use of animals and procedures were approved by the Ethical Committee for Animal Welfare (ECD, Ethische Commissie Dierenwelzijn) of KULeuven and IMEC. Timely pregnant FVBAtomic force microscopyConcentrated protofibrils or fibrils of Ab42CC, Ab42, or Ab40 were diluted to 0.5 to 1 mM in 20 mM sodium phosphate buffer at pH 7.2 with 50 mM NaCl, and 5 mL solutions were loaded onto freshly cleaved mica. After 1 to 2 min, the mica surface was briefly washed with 100 mL deionized water and air-dried. The samples were imaged immediately in AC-mode using a Cypher AFM instrument (Asylum Research, USA) equipped with NSC36/ Si3N4/AlBs three-lever probes (mMasch). The probes had nominal spring constants of 0.6 to 1.8 N/m and driving frequencies of 75 to 155 kHz. To determine protofibril length distributions, a number of images covering 1 to 2 mm2 surfaces were scanned and the lengths of particles were measured using a freehand tool in the MFP-3DTM offline section analysis software. The same tool was used to measure cross sections of particles.Analytical ultrace.