*2.4. Immunofluorescence Labelling of Myocardial Cx43, Cadherin, β-Catenin and Quantitative Image Analysis*

Cryostat sections from the left ventricle were used for the in situ immunodetection of Cx43, cadherin and β-catenin. The tissue sections were fixed in ice-cold methanol, permeabilized in 0.3% Triton X-100, washed in phosphate-buffered saline (PBS) and blocked with 1% bovine serum. The sections were incubated overnight with primary anti-Cx43 antibody (1:500, MAB3068, CHEMICON International, Inc., Temecula, CA, USA), anticadherin (1:300, sc-7939, Santa Cruz, Dallas, TX, USA) and with anti-β-catenin (1:250, sc-7963, Santa Cruz, Dallas, TX, USA) at 4 ◦C, washed in PBS and subsequently incubated

for one and a half hours with secondary antibodies conjugated with anti-mouse FITCfluorescein isothiocyanate (1:500, Jackson Immuno Research Labs, West Grove, PA, USA) or with anti-rabbit Alexa Fluor 594 (1:500, Jackson Immuno Research Laboratory Labs, West Grove, PA, USA).

The immunostaining of Cx43, cadherin and β-catenin was examined using a Zeiss Apotome 2 microscope (Carl Zeiss, Jena, Germany), and digital images were used for the quantitative analysis (Image-Pro Plus). Ten randomly selected areas were examined per heart. The quantification of Cx43 located on lateral sides of the cardiomyocytes (as a marker of the pathophysiological topology of Cx43) was performed as previously described [22]. After manual delineation of terminal intercalated disc-related Cx43 immunolabeling, the difference between the total IOD and IOD of terminal Cx43 corresponded to lateral Cx43. It was expressed as a percentage calculated from the ratio of lateral topology divided by the total IOD.
