Amersham hybond p 0

Western blot was performed to observe the changes in Aβ assembly after injecting TDP-43 to the APP/PS1ΔE9 mice. The concentration of the extracellular-enriched fraction and Triton-soluble fraction were determined and adjusted to the same protein concentration (48 μg in 20 μL). The samples were mixed with 6x loading buffer and heated in 95 °C for 10 min, then loaded into a 4–15% Tris-glycine gel and resolved at 100 V for 120 min. The separated proteins were transferred to a PVDF membrane (Amersham Hybond P 0.45, GE healthcare, Chicago, Illinois, USA) and blocked with 5% non-fat milk. The mouse monoclonal 6E10 antibody (1:5000, SIG-39320, BioLegend, San Diego, CA, USA) and mouse monoclonal 4G8 antibody (1:5000, SIG-39220, BioLegend, San Diego, CA, USA) were mixed and used for Aβ detection. A mouse monoclonal GAPDH antibody (1:5000, Proteintech, Rosemont, IL, USA) was used for loading control. The secondary antibody used for 6E10/4G8 mixture is goat anti-mouse IgG-HRP (GTX213111-01, Genetex, CA, USA) and for GAPDH is goat anti-rabbit IgG-HRP (GTX213110-01, Genetex, CA, USA). Immobilon Western Chemiluminescent HRP substrate (Merck, Darmstadt, Germany) was used for developing. The detection was carried out with Imagequant LAS4000 system (GE Healthcare, Life sciences, Hungary) and analyzed using ImageJ (NIH, Bethesda, MD, USA).