Brains were removed and snap-frozen in cooled isopentane. Frozen brains were cut into 10 μm thick sagittal sections using a cryostat (Leica) and mounted onto poly-L-lysine-coated www.selleckchem.com/products/VX-770.html glass coverslips. Brain sections were allowed to dry for at least 2 hr at room temperature and then washed with hybridoma serum-free media (H-SFM; Invitrogen) containing 1% FBS. Brain sections were then cultured with 5 × 105 BV2 cells in H-SFM containing 1% FBS for 18 hr at 37°C with 5% CO2 or 1.75 × 105 primary mouse microglial cells in H-SFM containing 1% FBS and 5 ng/ml GM-CSF (R&D Systems) for 60 hr at 37°C with 5%

CO2. Sections were then washed twice with PBS and either fixed with 4% paraformaldehyde for subsequent histology or placed in 6.25 M guanidinhydrochloride to extract Aβ for ELISAs (see below). Enzyme-linked immunosorbent assays were performed using Meso Scale technology (Meso Scale Discovery). Multiarray 96-well plates (Meso Scale Discovery) were coated with capture antibody 21D12 for total Aβ (Aβ13–28) or antibody 21F12 for Aβ42 (Aβ33–42). Plates were washed www.selleckchem.com/products/XAV-939.html and diluted samples and Aβ standards were added. Aβ was detected using biotinylated-3D6 antibody (Ab1–5) and SULFO-TAG streptavidin (Meso Scale Discovery). Plates were read on a Sector Imager 2400 (Meso Scale Discovery) and samples

were normalized to Aβ standards. All Aβ antibodies were provided by Elan Pharmaceuticals. Fibrillar Aβ1–42 was prepared by incubating synthetic monomers, diluted to a stock concentration of 1 μg/μl in PBS, overnight at 37°C. pH-sensitive beads were prepared by coupling 3 μm latex beads with CypHer5E mono N-hydroxysuccinimide (NHS) ester (GE Healthcare). Beads were washed extensively and diluted in PBS to a stock concentration of 2 × 104 beads/μl. Eight-week-old mice were then anesthetized with an inhaled isoflurane/oxygen mixture and 1 μl Aβ or pH-sensitive not beads were stereotaxically injected into the frontal cortex using the following coordinates from bregma: +1.9 μm anterior, +1.5 μm

lateral, and a depth of 1 μm with an injection speed of 0.2 μl/min. Forty-eight hours later, mice were perfused as described above and brains were prepared for histological analysis. Fixed sections were permeabilized with 0.1% Triton X-100 and 0.6% hydrogen peroxide. For Aβ histology, sections were blocked using a streptavidin and biotin blocking kit (Vector), and biotinylated 3D6 antibody was applied (1:8,000) overnight at 4°C. Primary antibody labeling was revealed using ABC kit (Vector) with diaminobenzidine (DAB; Sigma-Aldrich). For fluorescent double-immunolabeling, Iba-1 and 3D6 primary antibodies were detected with fluorophore-conjugated secondary antibodies (Alexa Fluor 488 and 555, respectively; Invitrogen). For studies with beclin 1+/− mice, coronal sections were cut at 50 μm for in vivo pH bead analysis using a freezing microtome (Leica).