Carl axio imager z2

Another group of animals under the same experimental conditions had their hearts extracted to make histological cross sections which included the left and right ventricular chambers. The sections were washed in a 0.9% NaCl solution and fixed in 10% formaldehyde and pH 7.4. Once the tissue was perfectly fixed, it was processed according to conventional histological procedures and stained by Masson’s trichrome at 16×. All representative microphotographs of the groups were taken from areas irrigated by the left anterior descending coronary artery, from the tip of the heart and from the anterior wall of the left ventricle, and approximately two-thirds anterior to the ventricular septum. Observations were performed at 16×.
Analysis of the marked histological sections was carried out with a Carl Zeiss light microscope (Carl Zeiss Axio Imager Z2, West Germany objective EC Plan-Neofluar 16×) and with an HP Z800 computer and an HP ZR30W screen. The photomicrographs were analyzed by densitometry using the SigmaScan Pro 5 Image Analysis software.

Immunofluorescence was performed on 4% formaldehyde/PBS fixed, 1% Triton-X/PBS treated cells using the following antibodies: Rad51 (Clone PC130, 1:200, Calbiochem) phospho-RPA pT21 (Cat. # ab109394, 1:500, Cell Signaling) γH2AX (Cat. #07–164, 1:500, EMD Millipore), and CyclinA2 (Clone CY-A1, 1:500, Sigma-Aldrich). Slides were scanned on the Carl Zeiss Axio Imager Z2 equipped with a CoolCube1 camera (Carl Zeiss, Thornwood, NY). Metafer 4 software (MetaSystems, Newton, MA) was used for automated quantification of Rad51, pRPA, γH2AX foci. More than 500 cells were quantified for every experimental replicate. Images were exported and processed on ISIS software (MetaSystems).

The histological sections of the left ventricle were done after the ventricle had been washed in 0.9% NaCl for 30 sec. and fixed by immersion in phosphate buffer with 10% formalin (pH 7.4) for 24 h. The sections were processed according to conventional histological procedures by hematoxylin-eosin stain. A Carl Zeiss light microscope (Carl Zeiss Axio Imager Z2, West Germany) with objective EC Plan-Neofluar 10x, with an HP Z800 computer and HP ZR30W screen, was used to analyze the histological sections. The photomicrographs were studied using densitometry with the SigmaScan Pro 5 Image Analysis software. The density values are expressed as pixel units.