Ps199

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.

Hippocampus and cortex was microdissected from frozen brains and processed to collect both soluble and insoluble extracts. Briefly, microdissected tissue was homogenized in TPER (ThermoFisher) and centrifuged at 12,000 RPM for 15 min. Supernatant was collected as the soluble fraction and the pellet was treated with 70 % formic acid and spun down at 25,000 rpm. The resulting supernatant was collected as the insoluble fraction. Insoluble protein samples were neutralized for Western blotting and further precipitated with trichloroacetic acid (TCA) when probing for insoluble tau. Protein samples were denatured at 95 °C for 15 min before being loaded onto 4-20 % TGX precast polyacrylamide gels (Bio-rad). Antibodies used for western blotting include: HT7 (1:1000), PS199 (Abcam; 1:1000), PS202 (Abcam; 1:1000), AT100 (ThermoFisher; 1:1000), AT270 (ThermoFisher; 1:1000), PHF1 (1:1000), 6E10 (1:1000), GFAP (1:1000). Mesoscale Discovery immunoassay kits (Mesoscale Diagnostics) were used for cytokine (K15048G) and Aβ38, 40, and 42 (K15199E) quantification of cortical samples following the manufacturers protocols. The proinflammatory MSD was able to detect levels of that were within the standard curve, whereas brain levels of IFN-γ, IL-12p70, CXCL1 and IL-4 were below the threshold of detection.