Anti c9

Hearts were removed after ischemia-reperfusion, fixed in 4% paraformaldehyde, embedded into OCT compound, quickly frozen at -80°C, and then processed for cryosectioning (5 µm thickness). Ten sections were prepared at 10 different transversal levels at the site of tissue necrosis, equally distributed from base to apex.
After the cryosections were fixed with acetone at 4°C for 30 min, the sections were stained with anti-Cx43 (1:200; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) and anti-C9 (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA), anti-Lc3-β (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) or anti-cathepsin D (1:50; Santa Cruz Biotechnology, Inc., Santa Cruz, Calif., USA) at D r a f t 4°C overnight. This procedure was followed by a 60 min incubation at room temperature with Alexa Fluor 488 goat anti-mouse IgG or tetramethylrhodamine goat anti-rabbit IgG second antibody. Hoechst 33342 (5 µg/mL, Molecular Probe) was used to label the nuclei. The sections were examined using a laser-scanning confocal microscope equipped with the FV10-ASW system (Olympus FV1000). The images were analyzed using Image-Pro Plus 5.0 software (Media Cybernetics Inc., Rockville, Md., USA)