Mitosox

Harvested organs were immediately placed in 10 mL of 10% (v/v) neutral buffered formalin (VWR, Radnor, PA, USA) and kept on ice until transferred to a 4 °C cold room, where organs were fixed overnight on a rocker. Organs were rinsed with 1× PBS, pH 7.4, for 10 min (3×) before embedding in paraffin or Tissue-Plus OCT (Thermo Fisher Scientific, Waltham, MA, USA). For histology, organs were cut in 6 µm serial sections using a CryoStar NX70 (Thermo Fisher Scientific, Waltham, MA, USA) and stained with MitoSox or H&E (Thermo Fisher Scientific, Waltham, MA, USA), or Trichrome (Polysciences, Warrington, PA, USA). Histological differences were quantified using ImageJ software. H&E and Trichrome images were collected by an Olympus VS120 virtual slide system (Olympus Life Sciences, Waltham, MA, USA). MitoSox images were collected with a Eclipse Ts2 fluorescent microscope (Nikon, Melville, NY, USA). Mean technical replicates (3–6 per parameter) were used to represent effects on a single heart. A minimum of three hearts were used for all quantified parameters. Protein quantification experiments used corresponding organs from seven different wildtype male, wildtype female, p66Shc^−/+ male, or p66She^−/+ female fish for a total of N = 7 per quantification.

H₂DCFDA, CellROX DeepRed and Mitosox were purchased from Invitrogen (Molecular Probes, Eugene, OR, USA). Whole mycelia and protoplasts were incubated with dyes in the growth medium for 10–15 min and washed in the medium 3 times according to the manufacturer’s instructions. Samples were observed and photographed under a Nikon fluorescence microscope using a filter with 495 nm excitation and 500–550 nm emission wavelengths (H₂DCFDA and Mitosox staining) or a filter with 510 nm excitation and 580 nm emission wavelengths (CellROX Deep Red staining).

Intracellular ROS was measured by MitoSOX (Thermo Fisher Scientific; Rockford, IL, USA) and 2’,7’-dichlorofluorescein diacetate (DCF) (Sigma, St Louis, MO, USA) as previously described.[21 (link)] At the end of reoxygenation, the cardiomyocytes were stained with 5 μM MitoSOX Red or 5 μM DCF to detect superoxide anion. Cells were washed and imaged using a Nikon-Eclipse80i confocal microscope with 561 nm and 488 nm excitation for MitoSOX Red and DCF respectively. The fluorescence intensity of MitoSOX Red and DCF were qualified with ImageJ software, as reported previously.[22 (link)] The data are presented as fold change in the median intensity of the fluorescence when compared with the respective controls.