Methoxy xo4

Mice were injected intraperitoneally with methoxy-X-O4 (Tocris) (10 mg/kg bodyweight), a fluorescent congo red derivate, in a DMSO/PBS mixture. After 3 h, hippocampi were collected and microglial cells were assessed as described previously [65 (link)]. Percentage of methoxy-XO-4 positive microglia were determined by flow cytometry using a FACS Canto II (BD Bioscience) and analyzed with FlowJo software (Tree Star).

A Nikon A1R MP microscope and a titanium–sapphire laser (Chameleon Ultra, Coherent, Santa Clara, CA) were used for mouse brain in vivo imaging. The same mice were imaged along three different time points (Appendix Fig S5), before (base), 2 and 10 days after LPS injection. The brain vasculature was used as a reference point to longitudinally image the same brain areas. Importantly, none of the mice died during the imaging session.
During all imaging sessions, the laser power did not exceed 30 mW. Three‐dimensional Z‐stacks (40 μm length; 0.5 μm step between optical planes) were acquired using a Nikon 25× objective (1.1 NA). For visualization of blood vessels, 20 mg/kg body weight dextran red 70KDa (Sigma‐Aldrich, Darmstadt, Germany) was i.p. injected 30 min before the imaging session. To visualize Aβplaques, 10 mg/kg methoxy‐XO4 (Tocris Bioscience, Bristol, UK) in 50% DMSO/50% NaCl (0.9%), pH 12, was i.p. injected 3 h before the imaging session (Bolmont et al, 2008).
For quantitative analysis, two‐photon Z‐stacks were automatically reconstructed using a self‐customized python‐based script (Ativie et al, 2018). Reconstructions were visually checked using the ImageJ plugin “simple neurite tracer” (Appendix Fig S6). Each cell was individually extracted, and all the individual files were analyzed using the open source software L‐measure (Scorcioni et al, 2008).

APP23 mice were treated with SUS^+MB and were perfused with PBS and drop fixed in 4% paraformaldehyde in PBS. They were then cryoprotected in 30% sucrose in PBS and sectioned at 40 μm with a freezing sliding microtome. Microglia were immunostained with anti‐Iba1 antibody (Wako, JP 1:1000) followed by incubation with an anti‐rabbit secondary antibody AlexaFluor 568 conjugate (Invitrogen). Sections were then co‐stained with 10 μM methoxy‐XO4 (Tocris Bioscience) in PBS with 20% ethanol before cover‐slipping. Images were obtained with a spinning disk confocal microscope (Nikon Diskovery) with a 20× objective, acquiring z‐stacks through the entire depth of the section (Figure 1d).

Microglia were immunostained with anti-Iba1 antibody (Wako, JP 1:1,000) followed by incubation with an anti-rabbit secondary antibody AlexaFluor 568 conjugate (Invitrogen). Sections were then co-stained with 10 μM methoxy-XO4 (Tocris Bioscience) in PBS with 20% Ethanol before coverslipping. Images were obtained with a spinning disk confocal microscope (Nikon Diskovery) with a 20x objective acquiring z-stacks through the entire depth of the section. An experimenter blinded to experimental groups then manually outlined plaque areas and manually counted the number of microglia cell bodies within 20 μm of the perimeter of a plaque using Nikon NIS software. The total number of plaques analyzed was 179 total, 82 for the sham condition and 97 for the SUS condition. Three-four randomly chosen fields of view were captured from 2 to 4 sections from each mouse, resulting in a total number of between 14 and 23 plaques analyzed from each mouse.