Dm750 light microscope

The morphological changes of the algal cells were observed using a Leica DM750 light microscope (Leica Microsystems, Wetzlar, Germany) and photos were taken with a Leica ICC50 W camera (Leica Microsystems, Wetzlar, Germany).

The growth rate of fungal mycelia was measured on PDA. The macroscopic characteristics of pycnidia formation were observed [20 (link)] with a Leica S8AP0 stereomicroscope (Leica Microsystems, Wetzlar, Germany), whereas the microscopic characteristics of the conidia were examined with a Leica DM750 light microscope (Leica Microsystems, Wetzlar, Germany). The dimensions of the conidia were measured. The fungal cultures were then deposited in a culture collection, and the accession number was assigned.

The rabbit skin tissues were fixed with 4% paraformaldehyde (PFA) and embedded in paraffin. Subsequently, we transferred 5-μm sections, cut using a microtome, to ProbeOn Plus slides (Fisher Scientific, Pittsburg, PA, USA), and stained the slides with hematoxylin and eosin (H&E) and Masson’s trichrome. We also stained the sections with Sirius Red (Sigma, Steinheim, Germany) at room temperature for 1 h. After staining, the sections were hydrated with a series of graded concentrations of ethanol, cleared with xylene, and mounted by using neutral resin. All slides were viewed using a Leica DM750 light microscope with an ICC50 HD camera attached (Leica Microsystems Ltd, Switzerland).

The right testis tissues were sliced into small pieces (2 mm³) and then fixed in paraformaldehyde (4%) in phosphate buffer pH 7.2 for 2 h at 20 to 22°C. They were dehydrated in a graded series of alcohols. In order to improve infiltration, the samples were treated with a mixture of LR White (Electron Microscopy Sciences, FT Washington, PA) and 70% ethanol (2 : 1) (v: v) for 1 h at 20 to 22°C. The samples were then embedded in LR White and sectioned at 700 nm (0.7 microns) thickness by using a Leica EM UC7 ultramicrotome. Semithin sections were stained with 1% toluidine blue/borax (pH 8.4) for 2 min and observed under a Leica DM 750 light microscope [46 (link)–48 (link), 59 ]. Spermatogenesis and testicular injury were evaluated using Johnsen's mean testicular biopsy score criteria. A score of 1–10 was assigned to each tubule cross section (n = 160) according to the range from no cells to complete spermatogenesis. Complete spermatogenesis with many spermatozoa present is evaluated as score 10. The total Johnsen score is then determined by dividing the total score by the number of evaluated tubules. At the cellular level, three pathological viewpoints (spermatogonial swelling, cytoplasmic vacuolation, and deformation of cellular architecture) were estimated on a semiquantitative scale and indicated as low (+), moderate (++), and high (+++) according to their degrees [60 (link)].

Tissue samples were fixed in 10% solution of neutral formalin in phosphate buffer (pH = 7.4) for 24 h, then treated with ethanol solutions of increasing concentrations (10%, 30%, 50%, 80% and 99%) and embedded in paraffin. Paraffin sections (5 μm thick) were dyed with hematoxylin and eosin (Bio-Optica, Milan, Italy). Microscopic analysis and registration of images were performed with the use of a Leica DM750 light microscope (Wetzlar, Germany).