Tr 300 ag

HEK293 cells were grown on poly-l-lysine (NeuVitro) coated glass coverslips for 24–48 h in 12-well plates before any treatment. At the end of the experiment, media was aspirated and cells were directly fixed in 4% formaldehyde (w/v) (Thermo, Methanol-free) in PBS for 10 min at RT, washed once with PBS and permeabilized in 0.1% Triton X-100/PBS for 5 min. Samples were blocked using 5% low-fat dry milk dissolved in 0.1% Triton X-100/PBS for 1 h at RT, followed by incubation with primary antibodies in blocking solution and fluorescently labelled secondary antibodies in PBS. Nuclei were counterstained with DAPI. For amyloid staining, after fixation and permeabilization, cells were incubated with Amylo-Glo (Biosensis TR-300-AG) at a dilution of 1:200/PBS with gentle shaking followed by washing twice with PBS. Cells were not counterstained with DAPI. Coverslips were mounted in fluorescent mounting medium (Dako) on glass slides and stored at 4 °C until imaging.

Fluorescent immunolabeling was performed using a standard indirect technique as described previously [22 (link)]. Brain sections were stained with primary antibodies against: ionized calcium binding adaptor molecule 1 (IBA1; 1:1000; 019-19741, Wako and ab5076, Abcam), CD68 (1:200; BioRad) glial fibrillary protein (GFAP; 1:1000; Abcam), NeuN (1:1000; Millipore), OLIG2 (1:200; Abcam), Aβ1–16 (6E10; 1:1000; Biolegend), and anti-lysosomal associated membrane protein 1 (LAMP1; 1:200; Santa Cruz Biotechnologies). For Amylo-Glo staining (TR-300-AG; Biosensis), tissue sections were washed in 70% ethanol 1 × 5 min, followed by a 1 × 2 min wash in distilled water. Sections were then placed in a 1% Amylo-Glo solution for 1 × 10 min then washed with 0.9% saline for 1 × 5 min and distilled water for 1 × 15 s before continuing fluorescent immunolabelling. For Thioflavin-S (Thio-S) staining, tissue sections were placed for 1 × 10 min incubation in 0.5% Thio-S (1892; Sigma-Aldrich) diluted in 50% ethanol. Sections were then washed 2 × 5 min each in 50% ethanol and one 10-min wash in 1xPBS before continuing with fluorescent immunolabelling.
High resolution fluorescent images were obtained using a Leica TCS SPE-II confocal microscope and LAS-X software. For confocal imaging, one field of view (FOV) per brain region was captured per mouse unless otherwise indicated.