Anti app

All the animal procedures were performed in accordance with University of Wisconsin guidelines. For in vivo injection, 1.5 μl of 1:2 AAV9 mixture of AAV9-APP-sgRNA:GFP (or AAV9-GFP) and AAV9-Cas9 was injected into the dentate gyrus (−2.0, ± 1.6, −1.9) of 8-week old male C57BL/6 mice (either sex)^{41 (link)}. Two-weeks after surgery, the mice were sacrificed by trans-cardiac perfusion of saline, followed by 4% PFA. The brains were dissected, post-fixed with 4% PFA overnight, immersed in 30% sucrose until saturation, and sectioned at 40 μm. Sections were immunostained with following antibodies: anti-HA (1:1000, 16B12), anti-GFP (1:1000, Invitrogen) and anti-APP (1:200, Y188). Images were acquired using Zeiss LSM800 confocal microscope. Average intensities of APP staining in cell bodies was quantified using Metamorph.

Rehydrated sections were treated with 1% H₂O₂ to block endogenous peroxidase activity, followed by citrate treatment as described above. Sections were washed with the addition of 0.1% Tween to the PBS solution, were blocked with 10% normal horse serum and incubated overnight with anti‐APP (Invitrogen 13–0200, 1:100) at 4°C. Sections were then incubated with biotinylated horse antimouse (1:200) for 45 min. The antigen was visualized as above. Following this, sections were then counterstained in Harris' haematoxylin (VWR, Dublin, Ireland) and dehydrated before coverslipping with DPX.
Immunohistochemically labelled sections were captured at 40×. Four sections of PrTN from 3–4 animals from each group were analysed as follows: pictures were processed to remove the Harris' haematoxylin counterstaining using the separate blue on image tool of Cell A software and saved as a new picture; the resulting pictures were opened in ImageJ and thresholded such that only APP‐positive labelled spheroids had sufficient transmittance to be retained. Spheroids were then assessed using the ‘analyse particles’ function using predefined parameters (size: 5–500 and circularity: 0.7–1). The mask tool allowed depiction of the selected particles for analysis. All samples were photographed on the same light and colour settings to minimize any influences of microscopy and camera settings.