S0063

To evaluate mitochondrial transmembrane potential (Δψm), the cells were stained with tetramethylrhodamine ethyl ester (TMRE, AAT Bioquest Inc. Sunnyvale, CA, USA) for 20 min at 37 °C, or with JC-1 (Mitochondrial Membrane Potential Assay Kit; C2006, Beyotime Inc., Shanghai, China). ROS production in living cells was assessed by dihydroethidium (DHE) staining (S0063, Beyotime Inc., Shanghai, China). To detect the production of mitochondrial superoxide, the cells were stained with MitoSOX (M36008, ThermoFisher Scientific, Waltham, MA, USA). Images were taken with an Olympus FluoView FV1000 fluorescence microscope (Olympus Optical Co., Ltd., Takachiho, Japan). Results were compiled from 20–45 cells from three or more independent experiments, and the same microscope settings were used for all treatments.

ROS levels were measured using a DHE kit (s0063, Beyotime), according to previous studies (Song et al., 2016 (link)). Briefly, mouse eyeballs were fixed and then OCT-embedded. Frozen sections (5 μm) were incubated with DHE dye in a 37°C incubator for 30 min. DHE was incubated with a superoxide anion that results in conversion to the red fluorescent compound ethidium. Fluorescence microscopy under the same exposure conditions was used to observe retinal layer ROS content. In retinal sections, the percentage of the ROS area stained with red fluorescence was normalized to the total area examined and quantified with ImageJ 4.0 analysis software.

The ROS levels were measured using the fluorescent probe, dihydroethidium (Beyotime, S0063, Shanghai, China). dihydroethidium (DHE), a fluorescent probe, could be dehydrogenated with reactive oxygen species (ROS) to produce ethidium, and then ethidium could bind to RNA or DNA to produce red fluorescence. The intensity of red fluorescence is proportional to the ROS levels. Thus, the detection of red fluorescence could determine the ROS level. Following behavioral tests, mice were perfused transcardially with PBS under anesthesia, and then the brains were removed and frozen rapidly. Subsequently, the brains were sliced into 20-μm slices in a freezing microtome (Leica, CM1900, Wetzlar, Germany). Referring to published studies, frozen hippocampal sections were incubated with 5 μM dihydroethidium at 37 °C for 30 min [54 (link),55 (link)]. Fluorescently labeled samples were imaged with a CaiZeiss confocal microscope (CaiZeiss, LSM800, Wetzlar, Germany). Specifically, the objective selected was Plan-Apochromat 20 × /0.80 Ph 2 M27, the laser power was 561 nm 0.60%, the detector gain was 643 V, and the field’s width of vision was 638.9 μm. In addition, three slides were used for analysis per mouse.

The detection kits of malondialdehyde (MDA, A003-1-2) and glutathione peroxidase (GSH-Px, A005-1-2) were purchased from Nanjing Jiancheng Bioengineering Institute. The activities of MDA and GSH-Px in serum were evaluated following the manufacturer's instructions, respectively. As for measurement of reactive oxygen species (ROS), the samples of the left ventricular myocardium were incubated with the DHE (5 μmol/L, S0063, Beyotime) at 37°C for 30 min, then washed with PBS for 3 times. The fluorescence intensity was examined using a confocal scanning microscope (LSM700, Zeiss, USA), and all images were analyzed using Image-Pro Plus software. And for each slice, 3 fields were randomly obtained for quantification.

Superoxide levels were measured by monitoring the fluorescence of dihydroethidium (S0063, Beyotime, Haimen, China)‐stained cells (lex = 300 nm, lem = 610 nm; Erhardt & Cooper, 1996) following the manufacturer's instruction. Briefly, cells were harvested from quadruplicate cultures, resuspended, incubated for 30 min in the dark and washed twice with PBS solution, and fluorescence was recorded. Whole‐mount fluorescent DHE staining was conducted in mice and chicken embryos at E10.5 and E4, respectively. Embryos were incubated with DHE (1:1,000) in serum‐free Iscove's modified Dulbecco's medium for 30 min at 37°C. After incubation with DHE, embryos were washed with PBS 3 times and examined immediately using a fluorescence microscope. Image analysis was performed blinded. All superoxide levels were expressed as relative levels to controls, which were arbitrarily set at 100%.

Frozen sections of the hippocampal tissue from mice were obtained. TUNEL (C1086, Beyotime), dihydroethidium (DHE) (S0063, Beyotime), and Iba-1 antibody (1:200, sc-32725, mouse monoclonal, Santa Cruz) were applied after washing with PBS three times. The cells were then counterstained with DAPI and examined under an inverted microscope (IX51, Olympus, Japan). TUNEL and DHE were performed according to the manufacturer’s instructions.

The production of reactive oxygen species was observed by performing DHE fluorescence staining (S0063, Beyotime, Shanghai, China) and measured as previously described [21 (link)]. Simply, H9c2 cells, which were transfected with si-Foxp1 or si-NC, were stained using DHE (10 μM) in the dark (30 min/37°C). Red DHE fluorescence was detected with the Olympus IX51 microscope (Olympus, Tokyo, Japan).