Axio vision le rel 4

Randomly chosen histological heart sections of four horizontal infarct levels were stained with Fast Green FCF (Sigma-Aldrich, Saint Louis, Missouri, USA) and Sirius Red (Chroma Waldeck GmbH & Co. KG, Münster, Germany) assessing tissue localization and distribution of connective fibers. Two contiguous levels of the heart (n = 6 for each group) which represent the major infarction ratio were quantitatively estimated using computer aided image analysis (AxioVision LE Rel.4.5 software, Carl Zeiss AG, Oberkochen, Germany). To evaluate leukocytes infiltration area 48 h after the second ligation, the two contiguous levels of the heart which represent the major infarct ratio were stained with Hematoxylin (Merck, Darmstadt, Germany) and Eosin (Thermo Shandon Ltd., Runcorn, UK) and representative images were taken using computerized planimetry (AxioVision LE Rel.4.5 software).

Tissue samples from the colon were rinsed with 0.9% NaCl, fixed in Bouin’s solution and embedded in paraffin blocks. The samples were then sliced into 5-μm sections and stained with haematoxylin and eosin. Crypt depth and myenteron thickness were measured using a Zeiss Axio Star Plus (Carl Zeiss, Göttingen, Germany) light microscope and Axio Vision LE Rel. 4.5 (Carl Zeiss, 2002–2005) image analysis software. At least 30 measurements of each parameter were performed per one sample and individual means for each piglet were calculated.

19–21-month-old +/N and N/N mice and age matched littermate control animals were fixed by intracardiac perfusion with 1% paraformaldehyde (PFA) plus 1% glutaraldehyde containing 0.2 M cacodylate buffer. Eyes were enucleated and fixed overnight in Karnovsky's buffer (2.5% glutaraldehyde, 2.0% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.2), washed with 0.2 M cacodylate buffer pH 7.4 and post fixed for 2 h in 1% osmium tetroxide and embedded in EPON (Serva, Heidelberg, Germany) after dehydration. Semi-thin sections (1 μm) from the central retina were cut along the vertical meridian of the eye at the optic nerve head (ONH) and counterstained with Methylene Blue and viewed on a Zeiss Axioskop-2 microscope using the AxioVision LE Rel. 4.5. software. Quantification of whole retinal thickness was assessed by dividing retinal areas into ten sections anterior and posterior of the ONH. Ultra-thin sections (50–80 nm) were contrasted with 4% uranyl acetate in 50% EtOH and 2% lead citrate in 1 M NaOH and viewed with an electron microscope (EM 902, Zeiss).

Paraffin-embedded caecal and colonic tissue samples were cut into 5-µm sections using a rotary microtome. Subsequently, the slides were deparaffinised with xylene, hydrated, and finally stained with haematoxylin and eosin. Crypt depth and myenteron thickness (15 measurements per slide, 2 slides per sample) were determined using a Zeiss Axio Star Plus light microscope (Carl Zeiss, Göttingen, Germany) and Axio Vision LE Rel. 4.5 image analysis software (Carl Zeiss, 2002–2005).