Dp72 colour digital camera

In situ (O₂^·-) production was detected by fluorescent probe dihydroethidium (DHE, Sigma-Aldrich, St. Louis, MO, USA) staining as described 25 (link). Briefly, the frozen sections of LV tissue (5 μm) from Rap1^+/+ and Rap1^-/- mice (male, 12 months old) were incubated with DHE (10 μM) at 37 ºC for 30 min. Fluorescence images were obtained using a BX41 microscope equipped with a DP72 colour digital camera (Olympus). The fluorescence intensity of DHE-labelled positive staining was quantified with Image J software in each of five randomly selected fields.

To analyse cardiomyocyte size, the LV adjacent sections from Rap1^+/+ and Rap1^-/- mice (male, 20 months old) were stained with AlexaFluor-594 conjugated wheat germ agglutinin (WGA, Invitrogen, Carlsbad, CA, USA) 23 (link). Briefly, myocardial slides were de-waxed, rehydrated, and boiled in sodium citrate buffer (10 mM) for 10 min before blocking with 1% (w/v) BSA in phosphate-buffered saline (PBS) for 1 h. The sections were then incubated in WGA (10 µg/mL in PBS) for 1 h in the dark at room temperature. Images of the slides, from five random separate fields, were taken using a BX41 microscope equipped with a DP72 colour digital camera (Olympus, Tokyo, Japan). The average cardiomyocyte size was quantified by measuring cardiomyocyte area (in µm²) from those photos with Image J software (National Institutes of Health, Bethesda, MD, USA).