A2547

Thioflavin S staining was carried out according to previously described procedures^{47 (link)}. We used SphK1 (rabbit, 1:50, Abgent, 8877), NeuN (mouse, 1:200, Millipore, MAB 377), 6E10 (mouse, 1:100, Signet, SIG39300), anti-20G10 (mouse, 1:1000, provided by D.R. Howlett, GlaxoSmithKline, Harlow, Essex, UK) for Aβ42, anti-G30 (rabbit, 1:1000, provided by D.R. Howlett) for Aβ40, SMA (mouse, 1:400, Sigma-Aldrich, A2547), AT8 (mouse, 1:500, Thermo Fisher Scientific, MN1020), MAP2 (chicken, 1:2000, Abcam, ab5392), Synaptophysin (rabbit, 1:100, Abcam, ab32127), Synapsin1 (rabbit, 1:500, Synaptic systems, 106 103), PSD95 (mouse, 1:100, Millipore, MAB1596), Iba1 (rabbit, 1:500, Wako, 019-19941), GFAP (rabbit, 1:500, Dako, N1506), S100 (rabbit, 1:200, Dako, Z0311), SOX2 (mouse, 1:100, R&D Systems, MAB2018), COX2 (rabbit, 1:50, Santacruz, SC7951), LOX-15 (rabbit, 1:100, Cayman chemical, 160704) and Lamp1 (mouse, 1:200, Abcam, ab24170). The sections were analyzed with a laser-scanning confocal microscope (FV1000; Olympus) or with a BX51 microscope (Olympus). MetaMorph software (Molecular Devices) was used for quantification. Three-dimensional reconstruction of microglia was recorded and analyzed using IMARIS software (Bitplane)^{48 (link)}.

PASMCs were plated and grown on glass slides until desired confluency, usually 70%–80%. Cells were treated with ezrin phosphorylation inhibitor NSC668394 (0.1 or 0.5 µM; Millipore) or vehicle (DMSO) and placed in the hypoxic chamber at 4% O₂ for 24 h. Cells were then washed, fixed with 4% formalin, and permeabilized with acetone. PASMCs were blocked and incubated with antibodies against SMA (Sigma, A2547, 1:500) and NHE1 (Thermo Fisher, PA5 115917 1:100) followed by incubation with fluorescent-conjugated secondary antibody (Cy5 goat anti-mouse for SMA and FITC goat anti-rabbit for NHE1, Life Technologies, Carlsbad, CA, United States) and DAPI nuclear counterstain. Images were obtained using a microscope with fluorescence objectives (Olympus I × 51, Center Valley, PA, United States) by an investigator blinded to treatment groups.

Thioflavin S (Sigma-Aldrich) staining was carried out according to previously described procedures^{31 (link)}. We used 6E10 (mouse, 1:100, Signet, SIG39300), anti-20G10 (mouse, 1:1000, provided by D. R. Howlett, GlaxoSmithKline, Harlow, Essex, UK) for Aβ42, anti-G30 (rabbit, 1:1000, provided by D. R. Howlett) for Aβ40, SMA (mouse, 1:400, Sigma-Aldrich, A2547), AT8 (mouse, 1:500, Thermo Fisher Scientific, MN1020), Caspase-3 (mouse, 1:200, Novus Biologicals, NB100-56708), ac-S565 (rabbit, 1:100), COX2 (mouse, 1:250, Thermo Fisher Scientific, 35-8200), Iba1 (rabbit, 1:500, Wako, 019-19941 and goat, Abcam, ab5076), NeuN (mouse, 1:500, Millipore, MAB177), GFAP (rabbit, 1:500, Dako, N1506 and chicken, 1:500, Abcam, ab4674), and Lamp1 (mouse, 1:200, Abcam, ab24170). The sections were analyzed with a laser-scanning confocal microscope (FV3000; Olympus) or with a BX51 microscope (Olympus). MetaMorph software (Molecular Devices) was used to quantification. Three-dimensional reconstruction of microglia was recorded and analyzed using IMARIS software (Bitplane)^{11 (link)}. For the analysis of age-dependent microglial COX2⁺ CX3CR1⁺ cells in WT and APP/PS1 mice, COX2 (rabbit, 1:100, Abcam, ab15191) and CX3CR1 (mouse, 1:100, BioLegend, 149008) antibody were used and the images were acquired on Alexa 488 and APC Channel with performed using Operetta CLS and Analysis Software 4.5 (Perkin-Elmer).