Planapo 40x 1.2 oil objective

NRVMs were prepared for staining by means of Cardiomyocyte Immunocytochemistry Kit (Thermo Fisher Scientific) following manufacturer’s instruction. Briefly, NRVMs were fixed with Fixative Solution for 30 min, permeabilized with Permeabilization Solution S for 15 min at room temperature and then blocked with Blocking Solution for 30 min. Mitochondria were stained using anti-TOM20 antibody (Santa Cruz; 1:500, rabbit) and sarcomeres were stained using anti-α-sarcomeric actinin antibody (Sigma; 1:500, mouse) over-night at 4°C. The day after, samples were incubated with Alexa Fluor 594 conjugated anti-rabbit (Life Technologies, 1:250) and Alexa Fluor 488 conjugated anti-mouse (Life Technologies, 1:250) for 1h at room temperature. Coverslips were mounted using NucBlue™ Fixed Cell ReadyProbes™ Reagent with DAPI to stain nuclei (Invitrogen). Images were collected at Zeiss LSM 700 confocal system equipped with a PlanApo 40x/1.2 oil objective, and an Argon laser used to excite the fluorophores at the appropriate wavelength. Images were collected at 2048×2048 pixels per image with a 100 Hz acquisition rate. Images were analyzed using the Fiji distribution of the Java-based image processing program ImageJ [38 (link)]. The experiments were performed both in normoxia and after 6h of anoxia followed by 30 min of reoxygenation.