Ab3754

Two weeks after AAV9 vector transfer of the reporter gene (ZsGreen) into rat myocardium, 6 μm cryosections were prepared from rat heart, lung and liver tissues, and examined for green fluorescence protein expression under fluorescence microscopy (Olympus BX-51 microscope, Olympus, Melville, NY, USA). Transfected HEK293 cells, isolated adult rat cardiomyocytes exposed to H/R, and fresh frozen ventricular sections from study groups were fixed for 10 min in 4% formaldehyde (Beyotime Biotechnology, Shanghai, China), stained with primary antibodies targeting PIASy (sc-166706, sc-50348, Santa Cruz Biotechnology, Danvers, MA, USA) and Cav-3 (ab2912, Abcam, Cambridge, MA, USA), Na_v1.5 (#ASC-005, Alomone Lab, Jerusalem, Israel), and SUMO2/3 (ab3754, Abcam, Cambridge, MA, USA), followed by incubation with goat anti-mouse IgG secondary antibody (#4408, Santa Cruz Biotechnology, Danvers, MA, USA) or goat anti-rabbit IgG secondary antibody (#8889, Cell Signaling Technology, Danvers, MA, USA). Nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI, C1005, Beyotime Biotechnology) during secondary antibody incubations. Fresh ventricular tissues were paraffin-embedded, and 5 μm sections were stained with hematoxylin and eosin (H&E). An Olympus fluorescence microscope was used for imaging.