Quantification of Cardiac Connexin 43 Immunofluorescence

For Cx43 immunodetection, 10 µm thick cryosections of myocardial apex tissue were used. According to our previous publications [59 (link),61 (link)], cryosections were fixed in ice-cold methanol, permeabilized in 0.3% Triton X-100, blocked in solution of 1% bovine serum albumin and incubated with primary anti-Cx43 antibody (diluted 1:500, CHEMICON International, Inc., Temecula, CA, USA, #MAB 3068 and secondary antibody with FITC-fluorescein isothiocyanate (diluted 1:500, Jackson Immuno Research Labs, West Grove, PA, USA, #111-095-003). For actin filaments, visualization cryosections were stained with phalloidin (Sigma-Aldrich, St. Louis, MO, USA, #P 2141). Microscopic images were captured by Zeiss Apotome 2 microscope (Carl Zeiss, Jena, Germany). Quantification of Cx43 immunofluorescence signal was performed on a 15 randomly selected myocardial area (per heart) and expressed as total integral optical density per area (IOD) [62 (link)].