Dialux 20

Light microscopy images were taken with a Leica MZFLIII (Leica microsystems) using a Leica DFC 320 camera and a Leitz Dialux 20 equipped with an Olympus EOS 7D camera. For lignin staining, roots were treated with phloroglucinol (Merck, Darmstadt, Germany; 1 g phloroglucinol in 40 ml 92% ethanol) for about 2 min, transferred to concentrated hydrochloric acid and investigated immediately.

Serial sections (3 μm thick), deparaffinized through incubation in a stove at 60 °C (30 min) and then in xylol (10 min), were hydrated in a series of incubations (5 min) in a decreasing series of ethanol aqueous solutions (100%, 90% and 70% v/v) and water. For each animal, 4 sections were stained with Hematoxylin–Eosin (HE) (Hematoxylin H and Eosin Y 1% v/v alcoholic, Biognost, Zagreb, Croatia) to assess cardiomyocyte area, and another 4 with 0.1% w/v Picrosirius Red (Direct Red 80, Sigma Aldrich, St Louis, MO, USA), to assess myocardial fibrosis.
Sections were visualized by an experienced and blinded individual in an optical microscope (Leitz Wetzlar—Dialux 20, Wetzlar, Germany) equipped with a camera (Olympus XC30, Tokyo, Japan). A cross-sectional area of 50 orbicular cardiomyocytes was measured per animal using Cell B software (Olympus), and eight fields were analyzed with Image-Pro Plus 6 software (Media Cybernetics, Rockville, MD, USA); the ratio of fibrotic area to total tissue area, corresponding to the percentage of interstitial fibrosis, was calculated.

The right ventricle (RV), LV and lungs were fixed in formalin, dehydrated in ethanol, cleared in xylol and impregnated in paraffin. Five-micrometer slides were dewaxed, rehydrated, and stained with
Haematoxylin-Eosin (HE), to assess cardiomyocyte area, or Picrosirius Red, to assess myocardial fibrosis, and finally mounted with Entellan®. An optic microscope (Leitz Wetzlar – Dialux 20, Wetzlar, Germany), equipped with a photographic camera (Olympus XC30, Tokyo, Japan) was used to visualise and photograph the histological preparations. The area of 60 cardiomyocytes per animal was measured using Cell^B software (Olympus). To calculate the area of fibrosis eight fields per animal were photographed and analysed with Image-Pro Pus 6 software (Media Cybernetics, Rockville, USA). To assess pulmonary arteries remodelling, HE staining was used, and artery medial wall thickness (WT) was expressed as follows: %WT = [(Medial wall thickness × 2)/Arterial external diameter] × 100. Analysis was performed in a blind mode. We attributed a code to each sample and the correspondence to the experimental group was done after analysis of the results.