Mouse hearts were excised and immediately fixed in formalin (CON) or first incubated without perfusion in non-oxygenated PBS for 30 min (ISCHEMIA). Paraffin embedded sections were co-labeled with a rabbit antibody to Total Cx43 (Sigma-Aldrich, St. Louis, MO) and a mouse antibody, CT1, which recognizes Cx43 unphosphorylated on S364/365 (FHCRC, Seattle, WA). Overlay panel shows the Total Cx43 antibody labeled with Alexa 546-conjugated secondary antibody in red and CT1 labeled with Alexa 488-conjugated secondary antibody in green.