Fluorosave

Cells grown on cover glasses were fixed with 1% (wt/vol) PFA in the medium at RT for 10 min. In the case of sphere cultures, 2% (wt/vol) PFA was used for fixation. Cells were made permeable with 0.5% Triton X-100 in TBS for 20 min. The samples were blocked with 3% (wt/vol) BSA and 10% (vol/vol) goat serum in TBS containing 0.1% Triton X-100 (TBS-T) and incubated with primary antibodies in the Can Get Signal immunostain solution (TOYOBO) at RT for 2 h. After washes, the cells were incubated with fluorescence-labeled secondary antibodies for 1 h. After further washes, cells were incubated with fluorescence-labeled phalloidin (Molecular Probes) for 30 min and subsequently mounted using FluoroSave (EMD Millipore). Images of cells were obtained using a laser-scanning confocal microscope LSM710/780 (ZEISS) equipped with an αPlan-FLUAR 100×/1.45 oil lens or Plan-Apochromat 63×/1.40 oil lens (ZEISS) at RT. Z-stack images were taken at every 0.3 µm. For observation of the lateral views with higher resolution, images were obtained by the laser-scanning confocal microscope LSM880-Airyscan (ZEISS) equipped with an αPlan-FLUAR 100×/1.45 oil lens at RT. Z-stack images were taken at every 0.17 µm and subjected to Airyscan super-resolution mode processing. Images were processed using ZEN software (ZEISS) and Photoshop CS5 (Adobe Systems).

Mice were sacrificed with CO₂ and perfused with ice cold PBS (10 minutes at 100 mL/h), and the brain divided into two hemispheres as described above. One of the hemispheres was fixed in 4% PFA overnight and then stored in PBS containing 0.01% sodium azide at +4°C until sectioning on a vibratome. For sectioning, the hemispheres were embedded in UltraPure agarose (Invitrogen, Carlsbad, CA) and cut into 35 mm sections on a Vibratome Leica VT1000S. Antigen retrieval was performed using boiling citrate buffer (Sigma-Aldrich) at pH 6.0. The sections were then blocked in PBS with 0.3% Triton X-100 and 5% normal donkey serum for 1h at RT and incubated in primary antibody at 4°C overnight. The following day sections were washed with PBS and incubated with secondary antibodies for 1 hour at RT. Nuclei were stained with DAPI and sections were mounted with Fluorosave (Merck Millipore). The following primary antibodies were used: mouse anti-β-amyloid 1–16 6E10 (BioLegend), rabbit anti-Iba1 (Wako-Chemicals, Neuss, Germany), goat anti-ApoE (Sigma-Aldrich). Secondary antibodies were: donkey anti-mouse Alexa 488 (Invitrogen A21202), donkey anti-rabbit Alexa 594 (Invitrogen A211207), donkey anti-goat Alexa 647 (Invitrogen A21447).