β amyloid

Splenocytes were isolated from spleens for flow cytometric analysis. Antibodies against the following surface markers were provided by: CD3 FITC (eBioscience™), CD4 PE (eBioscience™), CD8a Alexa Fluor 700 (eBioscience™), CD161 APC (eBioscience™) and CD19 PE-Cy5 (eBioscience™). Dead cells were stained by LIVE/DEAD® Fixable Blue Dead Cell Stain Kit (Life Technology). For immunohistochemical brain analyses, the left cerebral hemisphere was dissected and post-fixed in 4% paraformaldehyde in 0.1 M PBS for 2 days. Brains were cryoprotected by incubation in a 30% sucrose/0.1 M PBS solution. Sagittal brain sections were cut on a freezing microtome (Leica) and collected serially. Immunohistochemistry was performed on free-floating microtome-cut Sects. (10 μm in thickness). Sections were incubated with different antibodies: anti-mouse Iba1 (Polyclonal, 1:1000 Wako), anti-mouse CD68 (Clone FA-11, 1:200; BioRad), β-Amyloid (Clone 6E10, 1:1000; BioLegend), β-Amyloid 1–42 (polyclonal, 1:100; Millipore), anti-mouse GFAP (Clone GA-5, 1:100, Novus Biological) anti-human CD3 (Clone: CD3-12, 1:100, abcam) and anti-human Foxp3 (Clone 236A/E7, 1:100, Invitrogen). Appropriate secondary antibodies (Alexa Fluor 488, 594, or 647; Invitrogen) were used followed by incubation with DAPI.

Tissue sections stained by mRNA FISH and fixed primary neurons were washed with 1× PBS and blocked in 1× PBS supplemented with 5% (v/v) goat serum (Bio-Rad), 1% (wt/vol) BSA (Sigma) and 0.1% (v/v) Triton X-100 (Sigma) at RT for 1 h. Primary antibodies were diluted in blocking solution and applied to samples at 4°C overnight. Primary antibodies used in this study were: βAmyloid (BioLegend, 803001, RRID:AB_2564653, 1:200), GFAP (Sigma, G3893, RRID:AB_477010, 1:500), Homer (SynapticSystems, 160003, RRID:AB_887730, 1:500), Iba1 (Fujifilm Wako, 019-19741, RRID:AB_839504, 1:250), misfolded SOD1 (Médimabs, MM-0070-P, RRID:AB_10015296, 1:100), vGlut (MerckMillipore, AB5905, RRID:AB_2301751, 1:300); negative controls omitted the primary antibody. This was followed by 4× 10 min washes in 1× PBS at RT and subsequent application of suitable goat Alexa Fluor Plus secondary antibodies (488/546/647) diluted 1:500 in blocking solution at RT for 2 h. Samples were then washed 4× 10 min with 1× PBS at RT. Cryosections only were treated with 1× TrueBlack (Biotium) at RT for 30 s to quench autofluorescence caused by the accumulation of lipofuscin and other protein aggregates, followed by 2× washes with 1× PBS. Nuclei of samples were counterstained with DAPI (Sigma; shown in blue in all confocal images) at 1 μg/ml in PBS and samples were mounted using DAKO Fluorescence Mounting Medium (Agilent).