A1r plus confocal microscope

For confocal microscopy, images were scanned and acquired with a NIKON A1R Plus confocal microscope. Imaging parameters were kept constant in the same experiment. For plaque load and microglial area, 7 μm z-stacks consisting of 8 optical slices of 1 μm thickness were captured on a 20 × objective. Images were analyzed using the ImageJ (NIH) by adjusting threshold parameter to highlight all of the interested structures. The parameters were kept consistent throughout analysis. For quantifying the number of plaque-associated microglia, 9 μm z-stacks consisting of 10 optical slices of 1 μm thickness were captured on a 60 × objective. The number of microglia within a 25 µm radius of plaque was determined in a double-blinded manner.

Fluorescence resonance energy transfer (FRET) experiments were performed with the three channel sensitized emission method on living cells plated on a glass coverslip. Image acquisition was performed on a Nikon A1Rplus confocal microscope. A 405 nm laser line was preferred to a 458 nm one to limit acceptor bleed-through. The excitation light path was composed of: a 405/514/561/638 dichroic mirror, a 450/50 band pass in front of the first photomultiplier, a 50/50 beam splitter prior to a 525/50 band pass in front of the first GaASP and a 560 Long pass prior to a 525/50 band pass in front of the second GaASP. Sensitivity of the two Gasps and their gain/offset settings were adjusted with the 514 nm argon laser when exciting acceptor-alone transfected cells. Sensitivity of FRET and donor light paths were adjusted (same transmission power, different gain) in order to obtain a ratio FRET/Donor = 0.5. Finally, the 514 nm laser power was adjusted. All settings were kept for all acquisitions. A ×60/1.4 NA oil‐immersion objective was used, the confocal pinhole was set to three Airy resulting in quasi-wield field images. Image resolution was adjusted to a pixel size equal to 70 nm and a four lines average was achieved.

Human embryonic kidney 293 T cells (HEK 293 T) were transfected with pmCherry-N1, pmCherry-TREM1 or pmCherry-TREM2 plasmid. Twenty-four hours after transfection, cells were washed 3 times with DMEM and incubated with 1.0 μM FAM-labeled oAβ_1–42 for 2 h at 4 °C. After 3 washes with PBS, cells were fixed with 4% paraformaldehyde for 15 min. Cells were then stained with DAPI for 3 min and then washed twice with PBS for 15 min. Coverslips were mounted on the glass slide using antifade reagent (Thermo Fisher Scientific, P36935) and observed using NIKON A1R Plus confocal microscope.

Twelve-μm thick cryosections were washed in PBS for 15 min, permeabilized in 5% normal donkey serum and 0.2% Triton X-100 for 1 h, followed by 48 h’ incubation with Iba1 antibodies at 4 °C. The cryosections were washed with PBS for 30 min and treated with the Alexa-fluorophore-conjugated secondary antibodies for 2 h at RT. Sections were stained with DAPI (5 μg/mL), washed and mounted with anti-fade reagent. For confocal microscopy, 6 μm z-stacks (consisting of 13 optical slices of 0.5 μm thickness) were acquired using a NIKON A1R Plus confocal microscope.