Ix53 fluorescence microscope

ROS production was evaluated by DHE staining in vivo and DCFH-DA staining in vitro [38 (link), 40 (link)]. Briefly, cryosections of fresh heart samples or coverslips were stained with DHE (5 μmol/L) or DCFH-DA (5 μmol/L) in the dark at 37 °C for 30 min, and then were visualized in a blinded manner under an Olympus IX53fluorescence microscope. To further assess oxidative stress level, we measured the content of MDA, GSH, total SOD activity and NADPH oxidase activity in the myocardium or H9C2 cells according to our previous study by the commercially available kits [38 (link)]. Cell viability was determined using the CCK-8 assay kit according to the manufacturer’s protocol as described previously [9 (link), 38 (link)].

Paraffin sections were also used for immunofluorescence, and slides were incubated with primary antibodies against LC3 (CST, MA, USA). Slides were washed and incubated with fluorescence‐labelled secondary antibodies. The results were blindly calculated/section. After being washed 3 times with PBS, the H9C2 cells were visualized in a blinded manner under an Olympus IX53 fluorescence microscope.

Mice were anesthetized with pentobarbital sodium and transcardially perfused with PBS containing 5 U/ml heparin. Brains were dissected, cryosectioned with a thickness of 14–18 mm, and fixed in ice-cold acetone. Sections were blocked with 5% normal goat serum for 1 h at room temperature and incubated overnight with primary antibody goat anti-CD13 (R&D Systems; AF2335; 1:200) which was diluted in blocking solution at 4 °C. Sections were washed in PBS and incubated with the secondary antibody Alexa 568-conjugated donkey anti-goat (Invitrogen; A11057; 1:200). Further, sections were stained with Dylight 488-conjugated tomato lectin (DL-1174, 1:100, Vector Laboratories) and coverslipped with fluorescent mounting medium (Dako, Carpinteria, CA, USA) to visualize brain microvessel with a 510 confocal microscopy (Zeiss). For thioflavin-S staining, brain sections were stained for 10 min with 0.2% thioflavin-S (T1892, Sigma-Aldrich) diluted in PBS. After repeated wash with PBS, brain sections were imaged using an IX53 fluorescence microscope (Olympus). The quantification of images was analyzed using the Image J software.

The purified Exo were incubated with PKH26 (Sigma-Aldrich, St Louis, MO) for 5 min, rinsed with PBS, centrifuged at 120000 × g for 90 min, resuspended in basal medium, and then incubated with the target cells for 12 h at 37°C. Following incubation, the cells were rinsed twice with PBS and added with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) to stain the nucleus for 10 min. F-actin was stained with the help of phalloidin-fluorescein isothiocyanate (FITC). Afterwards, the stained cells were observed under an IX53 fluorescence microscope (Olympus, Tokyo, Japan).

Mice were anesthetized with chloral hydrate and perfused with 4% paraformaldehyde transcardially. The brain tissues were paraffin embedded and sectioned at the thickness of 5 μm. For thioflavin-S staining, we used 0.2% thioflavin-S (T1892, Sigma-Aldrich) to stain brain sections for 10 min. After the brain sections were washed with PBS for three times, the brain sections were photoed by an IX53 fluorescence microscope (Olympus). Image J software (GE Healthcare, USA) was employed to analyze the quantification of images.

NRVMs which were seeded in 6-well plates were exposed to HG for 24h. To assess the ROS level, the cells were incubated with DCFH-DA for 0.5h (37°C), and then were visualized in a blinded manner under an Olympus IX53fluorescence microscope. Cell count analysis (CCK-8; Dojindo Molecular Technologies, Rockville, MD, USA) was performed according to the manufacturer's protocol assays for cell viability.