Mouse monoclonal anti cx43 antibody

Left ventricular cryostat sections (n=5 per heart) were exposed to the mouse monoclonal anti-Cx43 antibody (Chemicon International, Inc.) at a dilution of 1:200 for 1 h at room temperature, followed by secondary FITC-conjugated goat anti-mouse antibody, as described previously (Lin et al. 2008) . Specificity of the immunoreaction was verified by incubation of the slices without primary antibody. Immunostained sections were examined using a fluorescence microscope (Axiostar, Carl Zeiss, Jena, Germany). Quantitative analysis was carried out as follows: a 2000 µm 2 square image was imported into NIH Image J software for analysis. The threshold for creating a binary image for counting was kept constant between images and was set to ensure that spots that represented Cx43 labeling would be counted without interference from background and the number of spots above background was counted automatically. The minimum detectable plaque size was 1 µm 2 . Four fields were analyzed for each set of measurements. The number of labeled gap junctions measured in each field was plotted as a frequency histogram.

Cryostat sections from the left ventricle were used for insitu immunodetection of Cx43 using mouse monoclonal anti-Cx43 antibody (Chemicon International, Inc.) and secondary FITC-conjugated goat anti-mouse antibody. Immunostained sections were examined using fluorescence microscope (Axiostar; Carl Zeiss, Jena, Germany) and images were acquired digitally for subsequent quantitative image analysis (Soft Imaging System, GmBh, Germany). Twenty randomly selected test areas were investigated per heart. From each test field a histogram of Cx43 fluorescence intensity was obtained and used for the calculation of Cx43 signal. The area of positive Cx43 labelling was defined as the number of pixels with the Cx43 signal intensity exceeding a threshold of 30 on the 0-255 gray scale. Total number of Cx43 positive pixels was expressed as integral optical density (IOD) per area. This parameter, expressed in arbitrary units, was compared between groups.

Cryostat sections from guinea pig heart atria were used for in situ immunodetection of cardiomyocyte distribution of Cx43. Following short fixation in freshly prepared paraphormaldehyde and washing in PBS, the sections were incubated overnight at 4 °C in primary, mouse monoclonal anti-Cx43 antibody (Chemicon International, Inc., USA, 1:200) and subsequently exposed for 1.5 h (in a dark place) to secondary FITCconjugated goat anti-mouse antibody (Chemicon International, Inc., USA, 1:200). Immunostained sections were examined using a fluorescence microscope (Axiostar; Carl Zeiss, Jena, Germany), and digitalized images were stored for subsequent quantitative image analysis (Soft Imaging System, GmBh, Germany). Twenty randomly selected myocardial areas were investigated per heart. The area of positive Cx43 indication was defined as the number of pixels with the Cx43 signal intensity exceeding a threshold of 30 on the 0-255 gray scale. The total number of Cx43 positive pixels was expressed as integral optical density (IOD) per area. The latter parameter, expressed in arbitrary units, was compared between young and old guinea pigs.