Shandon immu mount solution

Cell lines were plated onto #1.5 thickness, 12 mm round glass coverslips (Thermo Fisher Scientific) coated with Matrigel at ∼20% confluency and allowed to adhere overnight. Coverslips were washed with PBS and subsequently fixed in 4% paraformaldehyde in PBS at room temperature for 15 min. Coverslips were washed with PBS and incubated with 1:200 TOM20 (ProteinTech Group, 11802-1-AP) antibody prepared in PBS and 1% BSA for 2.5 h. Coverslips were then washed three times with PBS and incubated with 10 μg/ml Hoechst33342 counterstain (Invitrogen, H3570) and 1:200 Donkey Anti-Rabbit IgG (H + L) Cy5 (Jackson ImmunoResearch, 711–175–152) for 2 h. Coverslips were mounted onto Fisherbrand Superfrost Plus Microscope Slides (Thermo Fisher Scientific) using Shandon Immu-Mount solution (Thermo Fisher Scientific). Images were acquired through a Zeiss LSM880 AxioObserver Z1 confocal microscope with AiryScan using a 63x 1.4 NA oil objective. Images were processed using ZenBlue 3.2 software (Zeiss; https://www.zeiss.com/microscopy/en/products/software/zeiss-zen.html).

The brain sections were blocked for 1 h in blocking solution (10% horse serum, 0.3% Triton and 1XPBS), and then incubated with primary antibodies 6E10 (1:100, Biolegend) overnight at 4 °C. The following day, the slices were washed with PBS and then incubated for 1 h with secondary antibodies Alexa 555 (1:200, Invitrogen) at room temperature for 1 h and DAPI (1:5000, Sigma) for 3 min. Following this, the slices were carefully transferred to the slides and mounted with Shandon Immu-Mount™ solution from Thermo Scientific (Cat: 990402). 6E10-positive objects have been detected in the hippocampal circle area with a surface of 0.2 mm² with Zen Blue 2.5 (Zeiss, Oberkochen, Germany) software. Only dense-core Aβ plaques with a diameter of 10 ± 5 μm have been analyzed, and relative plaques density was calculated and presented as a bar-chart.