Axovision 4

Midventricular transversal sections of paraffin-embedded LV tissue (7 μm) were stained using the Sirius Red/Fast Green kit (Chondrex Inc., Redmons, WA, USA), according to the manufacturer’s instructions. After staining, sections were dehydrated in increasing concentrations of ethanol and mounted with a DPX mounting medium. Interstitial fibrosis was assessed in four randomly chosen 20× zoomed-in images using the Mirax Slide Scanner System (Carl Zeiss MicroImaging, Zaventem, Belgium). The area of collagen deposition was outlined, quantified using an automated image analysis program (Carl Zeiss, AxoVision 4.6, Zaventem, Belgium) and normalized to total surface area in %.

Transversal sections of 7 µm thick were obtained at cardiac midventricular level and stained using the Sirius Red/Fast Green kit (Chondrex). Fibrosis was assessed in all animals in four randomly chosen fields per section. The area of collagen deposition indicated by red staining was outlined and quantified using an automated image analysis program (Carl Zeiss, AxoVision 4.6, Zaventem, Belgium). Blood vessels were excluded. Total collagen deposition to the global cardiac area was calculated and expressed as percent collagen deposit.
Capillary density was quantified from histological sections by immunohistochemical staining for CD31 (SC-1506, Santa Cruz, 1/100). Capillaries were visualized by 3-3-diaminobezedine (DAB) and counterstained using hematoxylin. The amount of blood vessels were counted in 10 different fields per section and averaged. Data are expressed as amount of capillaries per µm².