Amylo glo rtd amyloid plaque stain reagent

Half brains were drop fixed in 4% (w/v) PFA for 48 h. The brains were cryoprotected in a 30% sucrose. Brains were sectioned coronally into 40 μm-thick slices on a freezing microtome (Leica SM 2010R) and stored in a solution of 0.05% NaN3 in 1× PBS as free-floating slices. For staining, tissue was blocked for 1 h in 1× PBS, 0.2% Triton X-100, and 10% goat serum. Immediately following blocking, brain sections were placed in primary antibodies diluted in 1× PBS and 1% goat serum and incubated overnight on a shaker at 4 °C. Samples were then incubated in conjugated secondary antibodies for 1 h followed by mounting on microscope slides. Sections were labeled with combinations of Amylo-Glo RTD Amyloid Plaque Stain Reagent (1:100; Biosensis TR-300-AG) (incubation for 20 min was needed before the addition of the primary antibodies), anti-GFP (1:500; Millipore Ab16901), anti-TagRFP (1:10,000; Kerafast EMU113), anti-Ku80 (1:250; Abcam ab79220), mounted with Fluoromount-G (SouthernBiotech). Additional samples were stained in anti-CXCR4 (1:100; MAB172 Clone 44716, R&D Systems), anti-CD9 (1:200; 312102, Biolegend), or anti-HLA-DRB1 (1:200; 14-9956-82, Invitrogen).

Cyclopiazonic acid (CPA) was from Sigma (St. Louis, MO, USA) (catalog no. C1530). Fura-2 AM was from Thermo Fisher Scientific (Waltham, MA, USA) (catalog no. F1221). Fluoro-Jade^® C was from Millipore (catalog no. AG325, lot 3170812). Amylo-Glo^® RTD™ Amyloid Plaque Stain Reagent was from Biosensis (Thebarton, Australia) (catalog no. T3-300-AG, lot BA01-30-300 AGTK120419). Mounting medium Entellan^® new was from Millipore (Burlington, MA, USA) (catalog no. 107961, lot HX42534561). Polyclonal rabbit monoclonal antibody against phospo-GluR1 Ser845 was from Millipore (catalog no. EPR2148; lot 2377032). Monoclonal antibody calcineurin (CaN) was from Sigma (St. Louis, MO, USA) (catalog no. C1956; lot 094M4820V). The housekeeping protein monoclonal mouse β-actin antibody was from Sigma (St. Louis, MO, USA) (catalog no. A54418, lot 122M4782). Secondary anti-rabbit and anti-mouse antibodies were conjugated with horseradish peroxidase (HRP; Sigma catalog no. A9169 lot 117M4808V and A9044 lot 055M4818V).

Following perfusion, one hemisphere from each mouse was postfixed in 4% paraformaldehyde for 48 h then stored in PBS + 0.05% sodium azide. Fixed half-brains were placed in 30% sucrose for at least 48 h before being cut in the coronal plane (40-μm sections) using a freezing sliding microtome. Brain sections were rinsed in PBS before blocking in PBS + 0.05% Triton-X with 10% goat serum for 1 h. First, samples were stained with Amylo-Glo™ RTD Amyloid Plaque Stain Reagent (Biosensis, Australia) for 15 min, washed three times, and then incubated in pS199 (Abcam, UK, 1:1000) and PHF-1 (gift from Dr. Peter Davis, 1:1000) phospho-tau primary antibodies at 4 °C overnight. The next day, sections were washed three times with PBS and placed in appropriate Alexa Fluor-conjugated secondary antibody solutions at room temperature for 1 h. Sections were rinsed three additional times, mounted onto slides, and coverslipped using Fluoromount-G. For confocal microscopy, immunofluorescent staining was performed on equivalent brain sections and imaged on the Olympus FX1200 confocal microscope. Tau tangles and β-amyloid plaques were visualized using Z-stack maximum-projection images taken through the entire depth of the section at 1-μm intervals.