Anti aβ 6e10 antibody

Nine month-old APMAP-KO/AD and their control APMAP-WT/AD mice were euthanized and the brain’s left hemispheres were collected, fixed for 48h in 4% paraformaldehyde diluted in PBS (Roche), cryoprotected for 24h in 30% sucrose diluted in PBS, and kept at -80°C in the cryopreservative matrix. Coronal hippocampal slices of 20μm thickness were obtained with a cryostat, and further processed in a free floating manner. First, slices were washed in PBS, boiled for 20 min in citrate buffer (Invitrogen) and cooled at room temperature. Next, blocking, binding of the primary anti-Aβ 6E10 antibody (Biolegend, London, United Kingdom) and binding of the secondary anti-mouse HRP antibody, were performed with the Immpress kit (Vector laboratories, Burlingame, CA, USA) according to manufacturer instructions. Next, staining was performed by using the SG-blue chromogen kit (Vector laboratories), according to manufacturer instructions. Finally, individual Aβ plaques were scored on slices mounted with fluorsave (Millipore, Schaffhausen, Switzerland) and analyzed by phase contrast microscopy (Zeiss, Feldbach, Switzerland).

Oligomeric assemblies of Aβ were derived from AD brain tissues (referred to as h‐iAβo) by following previously published protocol (Sengupta et al., 2015 (link)). Aβo was extracted from the PBS‐soluble fraction of AD brain homogenates using a co‐immunoprecipitation Kit (Thermo Fisher Scientific, USA) following the manufacturer's guidelines. Briefly, amine‐reactive resin was coupled with an anti‐Aβ 6E10 antibody (BioLegend) followed by incubation with the PBS‐soluble fraction of the AD brain homogenate. Bound proteins were eluted in 0.1 M glycine (pH 2.8), and the final pH was adjusted to 7.0 by adding 1 M Tris‐HCl (pH 8). The eluted fraction was subjected to buffer exchange and collected in sterile PBS. This fraction was further separated by size exclusion chromatography using the AKTA Explorer system fitted with a Superdex 200 Increase 10/300 gl Column. Degassed PBS was used as the mobile phase with a flow rate of 0.5 ml/min to collect the Aβo fraction. The total protein concentration was measured with a bicinchoninic acid protein assay (Pierce™ Micro BCA Kit, Thermo Fisher Scientific, USA). Human brain‐derived Aβo was characterized by Western blot analysis and atomic force microscopy.