Cell a software

Paraffin-embedded sections were placed on adhesive slides and stained with hematoxylin and eosin. Cardiomyocyte cross-sectional areas were calculated under a microscope using the Cell^A software (Olympus Soft Imaging Solutions GmbH, Germany).

After the blood sample collection, all rats were sacrificed by bleeding and the hearts were explanted. Pieces of the LV myocardial tissue were fixed in buffered paraformaldehyde solution (4%) and embedded in paraffin. Then, 5-μm thick sections were stained with hematoxylin and eosin. Cardiomyocyte cross-sectional areas were calculated on a microscope using the Cell^A software (Olympus Soft Imaging Solutions GmbH, Germany). An acid fuchsin orange G (AFOG)-stain was used to determine the extent of myocardial fibrosis. Four sections per heart were inspected under light microscopy and rated according to the following scoring system: grade 0 indicates normal tissue showing no fibrotic region; grade 1 indicates mild fibrosis; grade 2 indicates moderate fibrosis, and grade 3 indicates severe fibrosis. The histological evaluation was conducted by an analyst unaware of the experimental groups.

After the blood samples were collected, pieces of the left-ventricular myocardial tissue were collected for histopathology, fixed in buffered paraformaldehyde solution (4%), and embedded in paraffin. Then, 5 μm thick sections were placed on adhesive slides and stained with hematoxylin and eosin (H&E). Cardiomyocyte cross-sectional areas were calculated under a microscope using the Cell^∧A software (Olympus Soft Imaging Solutions GmbH, Germany). Masson's trichrome staining was performed to determine the extent of fibrosis. Four sections per heart were inspected under light microscopy and rated according to the following scoring system: grade 0 indicates normal tissue showing no fibrotic region, grade 1 indicates mild fibrosis, grade 2 indicates moderate fibrosis, and in grade 3 severe fibrosis is indicated. The histological evaluation was conducted by an investigator unaware of the treatment attribution of the animals.

Species identification in pure fungal cultures was carried out on the basis of macro- and micromorphological characteristics on Petri dishes and agar slants, respectively, with Sabouraud medium with the following composition (g dm⁻³): glucose—40; peptone—10; agar—20, distilled water—1 dm³, and in microcultures, which was described in detail in the study of Bohacz, et al. [26 (link)]. Macroscopic identification concerned the colony morphology of these fungi and their ability to produce pigment. Microscopic identification using an Olympus BX-41 microscope (Olympus, Tokyo, Japan) equipped with a CVIII4 camera (Olympus, Tokyo, Japan) and Cell-A software (Soft Imaging System GmbH, version (v.) 1.20, Münster, Germany) included hyphae structure, the presence and structure of micro- and macroconidia (anamorph), as well as the presence or absence of fruiting bodies (teleomorph). The species classification was based on the systematic keys listed in Bohacz, et al. [26 (link)].

AFOG staining was used to detect collagen fibers in the aortic tissue as described elsewhere [20 (link)]. The collagen content was determined by semi-quantitative morphometry scoring of the sections under a microscope using Cell^A software (Olympus Soft Imaging Solutions GmbH, Munster, Germany).

As previously reported [19 (link)], distal regions of the aortic segments were fixed in a buffered paraformaldehyde solution (4%) and embedded in paraffin. Then, 5 μm thick sections were placed on adhesive slides and stained with hematoxylin and eosin as described elsewhere [18 (link)]. The aortic morphometric measurements, including lumen cross-sectional area, intima-media area, and intima-media width, were determined under a microscope using Cell^A software (Olympus Soft Imaging Solutions GmbH, Germany), then normalized to body weight [20 (link)]. Additionally, the intima-media/lumen area ratio was calculated. The evaluation was conducted by an analyst blinded to the experimental groups.

According to previously described methods [12 (link)] we performed immunohistochemical staining for nitrotyrosine, a marker of nitrooxidative stress. Semiquantitative histomorphological assessment was performed based on the intensity and distribution of labelling using conventional microscopy. After initially evaluating all corresponding tissue sections (magnification 200x), the tissue section with the most intense labelling signals was used as a reference for maximum labelling intensity. Each specimen was characterized with the average of 4 adjacent fields. Nitrotyrosine levels were scored as follows: 0: complete absence of immunoreactivity, 1: weak area of staining, 2: intermediate staining, and 3: extensive staining. Using the Cell^∧A software (Olympus Soft Imaging Solutions GmbH, Germany), we measured the area of the objects in each class in each field, assigned an area score (1 ≤ 10% positive cells, 2 = 11–50% positive cells, 3 = 51–80% positive cells, and 4 ≥ 80% positive cells), and calculated an average score for the whole picture (intensity score multiplied by area score, 0–12). The evaluation was conducted by an investigator blinded to the experimental groups.