Fluoroshield containing dapi

To directly label plaques, brain cryosections were washed for 1 min each in 70% and 80% ethanol and then incubated with 1% thioflavin S in 80% ethanol for 15 min. Slides were then washed for 1 min each in 80% and 70% ethanol, rinsed in deionized water, and mounted with Fluoroshield containing DAPI (Sigma). For exosome injection, donor astrocytes were labeled with Vybrant CM-DiI (5 µL/mL DMEM) for 1 h followed by 40 µM Aβ25-35 for 3 days. DiI-labeled exosomes were isolated as described above, resuspended in sterile PBS, and injected into the brains 10-day-old 5XFAD mice. Mice were sacrificed 48 h later, and the brains fixed in 4% p-formaldehyde/PBS. For immunohistochemistry, brain cryosections were permeabilized with 0.2% Triton X-100 in PBS for 10 minutes. Amyloid plaques were labeled with 1 µg/mL Aβ1-42 ABfinity™ recombinant rabbit monoclonal antibody (Life Technologies) in PBS plus 0.5% BSA. The secondary antibody AlexaFluor488-donkey anti-rabbit IgG (Jackson Immunoresearch) used at 1µg/mL in PBS plus 0.5% BSA. Confocal fluorescence microscopy was performed with an LSM 510 confocal scanning microscope using LSM 510 Meta 3.2 software for image acquisition. Images obtained with secondary antibody only were used as negative controls representing the background intensity in a particular laser channel.

Formalin-fixed, paraffin-embedded hearts from sham and HF mice were sectioned at 5 μm thickness, deparaffinized, and rehydrated. Histological staining was performed as previously described.^{6 (link), 21 (link), 23 (link)} Masson’s trichrome was used to evaluate tissue fibrosis and Alexa Fluor 488–conjugated wheat germ agglutinin (Invitrogen) to assess myocyte area, as quantified from 5 to 6 high-power fields per section in non-infarcted myocardium using Metamorph software version 6.3r5 (Molecular Devices).
To evaluate for tissue abundance of CD4⁺ and CD8⁺ T-cells, heart sections were embedded in OCT compound (Tissue-Tek OCT), and then kept at −80°C until sectioning. Sections (7 μm thickness) were fixed with 4% paraformaldehyde in PBS, and labeled with either rat anti-mouse CD4 (Clone GK1.5; eBiosciences) or CD8 antibody (Clone 4SM15; eBiosciences). Goat anti-rat antibody conjugated with Alex Fluor 555 and 488 (Life Technologies) were used as secondary antibodies for CD4 and CD8, respectively, and fluoroshield-containing DAPI (Sigma-Aldrich) was used as the mounting medium. CD4⁺ or CD8⁺ cells were quantified using confocal microscopy from 5–6 sections and 3–4 mice from each group. All measurements were conducted in a blinded manner. Confocal microscopy was performed on an LSM710 microscope (Zeiss).