Dapi solution

For in situ visualization of the O₂⁻ and O₂⁻ derived oxidants, hydroethidine histochemistry was carried out at 3 days post LPS. Animals intraperitoneally received hydroethidine (1 mg/kg in PBS containing 1% dimethyl sulfoxide; Sigma) and were transcardially perfused with a saline solution containing 0.5% sodium nitrate and heparin (10 U/mL), and then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer. After fixation, the brain tissues were cut into 40-um slices using a sliding microtome. As described [30 (link),46 (link)], tissue sections were mounted on gelatin-coated slides, and the oxidized hydroethidine product, ethidium, was viewed by confocal microscope (Carl Zeiss), and then merged with DAPI solution (Vector Laboratories). To quantify, obtained images were analyzed by Image J (National Institutes of Health, Bethesda, MD, USA).

HeLa and HT-29 cells were grown onto glass coverslips in 6-well plates. After toxin exposure, cells were washed by PBS twice, fixed by 4% (wt./vol.) paraformaldehyde in PBS for 10 min, and permeabilized in 0.2% Triton X-100 in PBS for 10 min. The coverslips were mounted onto slides using mounting medium containing DAPI solution (Vector Laboratories, Vectashield), and the cells were examined by either LSM 510 laser-scanning confocal microscope (Zeiss) or TCS SP2 spectral confocal system (Leica).

The apoptotic bodies were stained using the 1 μg/ml DAPI solution (Vector Laboratories, USA) according to the manufacturer's instructions. Cells were treated with OJE fraction for 24 hr. After incubation, the cells were washed with cold PBS and then fixed in cold 4% paraformaldehyde for 30 min. Apoptotic bodies were dyed blue and fixed with mounting medium. After staining, cells were analyzed using fluorescence microscopy on AMG (Washington, USA).

C2C12 cells differentiated under various conditions were subjected to immunofluorescence staining. Cells were differentiated with or without activin A (50 ng/mL) for 5–7 days. Furthermore, cells transfected with micro RNA inhibitors were differentiated in the presence of activin A and T-MSC exosomes (5 μg/mL). All cells were cultured in a Lab-Tek Chamber Slide (Thermo Fisher Scientific). After washing with PBS, cells were fixed with 4% formaldehyde for 15 min and then incubated with primary antibodies against myogenin (Abcam, Cambridge, MA, USA) overnight at 4 °C. After that, washed slides were incubated with DyLight-conjugated anti-mouse IgG H&L (Abcam) for 1 h at room temperature in the dark. After washes, slides were mounted with DAPI solution (Vector Laboratories, Burlingame, CA, USA) and examined using a fluorescent microscope (Olympus, Tokyo, Japan).

Immunofluorescence was performed on 10-µm-thick sections. Following permeabilization with PBS containing 0.5% Triton X-100 for 15 min and blocking with 1% BSA in PBS for 60 min at room temperature, the sections were incubated with primary antibodies diluted in PBS containing 1% BSA and 1% Tween 20 at 4 °C overnight. Primary antibodies used in this study include 1:100 CD90 (Abcam #Ab225), 1:100 MYH6/7 (Abcam #Ab50967), 1:100 TNNT2 (Thermo Fisher #MA5–12960), 1:200 ACTN4 (Sigma-Aldrich #A7811), 1:100 GJA1 (Abcam #Ab11370), 1:100 human MYH7 (Abcam #Ab172967), and 1:100 human mitochondria (Abcam #Ab92824). After washing three times with 1% Tween 20 in PBS, the samples were incubated with secondary antibodies for 60 min at room temperature in the dark. Secondary antibodies used in this study include either 1:400 anti-mouse IgG Alexa Fluor 488 (Invitrogen #A10680) or 1:400 anti-rabbit IgG Alexa Fluor 647 (Invitrogen #A21245). After washing again with 1% Tween 20 in PBS, the sections were stained with DAPI solution (VectaShield) for nuclear staining and then mounted on slides. Imaging of heart sections was performed with a Laser Scanning Microscope LSM 880 NLO with Airyscan processing (Zeiss).