Ht15 1kt

Hearts were removed and fixed in 10% formalin for 48 hours, dehydrated with ethanol, and embedded in paraffin. Sections were cut at 7 µm, with 50 µm between each section, and Masson’s trichrome staining was performed (Sigma-Aldrich, HT15-1KT) according to the manufacturer’s instructions. Analysis and quantification of fibrosis was performed as previously described^{46 (link),50 (link)}, where 3 sections per heart were imaged on an Olympus BX51 microscope and quantified using ImageJ (NIH, Bethesda, MD).

After the occlusion protocol, the animals were sacrificed with pentobarbital (200 mg/kg, IV) administrated to the ewe through the jugular vein. Death was confirmed by the cessation of circulation and breathing in both ewes and fetuses. Muscular tissue surrounding the insertion area of the electrochemical sensors inserted was carefully excised; fixed for one week by immersion in 10% buffered formalin and embedded in paraffin for further histological analyses. Paraffin blocks were serially cut in 5 μm thick transverse sections with a microtome and standard hematoxylin/eosin (Mayer’s Hematoyxlin, Ref. 51275 Sigma-Aldrich, Saint Louis, MO, USA; Eosin, Ref. 1.15935.0100 Merk, Darmstadt, Germany) and masson trichrome staining (Ref. HT15-1KT, Sigma-Aldrich, Saint Louis, MO, USA) were performed. One representative section from each sample was examined under an optic microscope (CH-9435, type DFC425C, Leica Microsystems, Hospitalet de Llobregat, Spain). Tissue integrity and cell infiltration were analyzed with hematoxylin eosin staining, whereas masson trichome staining was used for the evaluation of fibrotic reaction. The design of this study is summarized in Figure 1.

Paraffin embedded sections of lungs and right ventricles were de-paraffinized, rehydrated and stained with Masson’s trichrome as per manufacturer’s instructions (Cat # HT15-1KT, Sigma-Aldrich). Fibrosis was qualitatively determined by examining slides under a light microscope (Model Leica DMI 4000B) at 40X magnification.

Slices from thoracic aortas were stained with hematoxylin and eosin and photographed with an Olympus BX41 microscope. The area and thickness of tunica media and the lumen area were calculated using Image J (Version 2.0.0-rc-69/1.52p. National Institutes of Health, Bethesda, MD). Additional slices were stained with Masson’s trichrome to determine qualitative total collagen content following the instructions from the Sigma-Aldrich kit HT15-1KT. Images were captured and processed with bright field of the microscope Zeiss Axio Observer Z1 using bright field and the 10× objective.

Hematoxylin and eosin staining (#051-06515, #131-09665, WAKO), Masson’s trichrome staining (HT15-1KT, Sigma), TUNEL assay, and immunofluorescent staining were performed on sections from formalinfixed, paraffin-embedded tissues. The myocyte cross-sectional area was measured from images captured from wheat germ agglutinin (WGA, W11261, Invitrogen)-stained sections. The outlines of 100-300 myocytes were traced in each section using NIH ImageJ. Lipofuscin was detected by autofluorescence in fluorescence microscopy. Lipofuscins appears as irregular granules that emit yellow-orange fluorescence between 500 and 640 nm, under any excitation wavelength ranging from 360 to 647 nm. SA-β-gal activity was determined using the Senescence β-Galactosidase Staining Kit (#9860S, Cell Signaling Technology) according to the manufacturer’s instructions. For mouse hearts, β-gal staining solution was used at a final pH of 5.0.