Lsm8800 airyscan confocal microscope

Cells were washed with PBS and fixed for 20 minutes with fresh 4% paraformaldehyde. Cells were permeabilized post fixation with 0.1% Triton X-100 for 10 min. See Key Resources Table for a list of primary and secondary antibodies. For CellROX images, cells were treated with 5 μM CellROX reagent for 45 minutes. Cell were washed 3 times with PBS and imaged live per the manufacturer’s instructions. TUNEL staining was performed using the In Situ Cell Death Detection Kit (Roche). Where cardiac tissue was stained, hearts were fixed for 24 hours in fresh 4% paraformaldehyde. Tissues were embedded in paraffin blocks and cut into 5 μm sections. Confocal images were acquired on a Zeiss LSM8800 Airyscan confocal microscope. Other fluorescence pictures were taken with a Keyence BZ-X700 microscope.

sEVs were stained with PHK26 per the manufacturer’s instructions and injected into C57BL/6 mice as specified in the Mouse treatments section. Mice were euthanized by cervical dislocation under isoflurane. The heart was perfused with ice cold PBS to remove blood. Cardiac tissue was separated into the left ventricle, right ventricle and atria and sliced into pieces ~10 mm thick. Pieces were mounted onto a microscope slide with Dako fluorescent mounting medium and PHK26 fluorescence was detected using a Zeiss LSM8800 Airyscan confocal microscope.