N1506

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).

Temporal cortex sections (7 μm) of human AD and control brain were prepared from formalin-fixed paraffin-embedded blocks. Sections were deparaffinized in xylene (Thermo Fisher Scientific, X/0250/17) and rehydrated in 99% (v/v) ethanol, followed by antigen retrieval in citrate buffer (0.01 M sodium citrate, pH 6) at 95° to 100°C for 5 min and 65°C for 12 min. Sections were cooled in water and rinsed in tris-buffered saline (TBS), and a hydrophobic wax boarder was applied using PAP pen (SLS, HIS0500). Sections were placed in blocking buffer [1:100 normal goat serum (Sigma-Aldrich, G9023) in TBS] followed by primary antibody incubation overnight: 6E10 (1:200, BioLegend 803002), ALDH1L1 (1:200, Antibodies-online ABIN1304519), CAII (1:100, Abcam ab124687), GFAP (1:500, Dako N1506 or Abcam ab4674), HSPB1 (1:200 Enzo ADI-SPA-803 or HSPB1 mouse Proteintech, 66767-1-Ig), IBA1 (1:200, Wako 019-19741), and MAP2 (1:500, Sigma-Aldrich 05-346 or GeneTex GTX82661). After fluorophore-coupled secondary antibody (Invitrogen) incubation at 1:250, sections were treated with 0.3% (w/v) Sudan Black (Acros Organics, 190160250) in 70% ethanol and mounted using Fluoromount-G mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen, 00-4959-52).