Dm3000 microscope

After extraction, the appearance of the testes and the state of the parenchyma on the cut were assessed and weighed (in grams). Then they were cut parallel to the sagittal plane every 2 mm, fixed in Bouin’s solution; after insertion (apparatus for histological tissue guiding, Leica Biosystems, Wetzlar, Germany), they were embedded in paraffin blocks, from which serial sections (2 µm thick) were prepared, dewaxed, dehydrated and stained with hematoxylin and eosin (H&E) for histological evaluation.
Morphological and morphometric analysis was carried out in 10 randomly selected fields of view of the microscope at ×100 and ×200 magnification in 4 random sections from each sample, moving the slides at equal intervals along the X and Y axes, using a semi-automatic image analyzer. Light microscopy was performed using a video microscopy system (Leica DM3000 microscope, Leica Biosystems, Wetzlar, Germany; DFC450 C camera; Platrun LG computer), and morphometric data were obtained using software for image processing and analysis (Leica Application Suite (LAS) Version 4.9.0) (Figure 1). The spermatogenic cycle of rats, which includes 14 consecutive stages, was assessed according to Chermont, Leblond and Messier (1959). Testicular assessment was carried out according to S. Johnsen criteria modified by De Kretser and A. Holstein (Table 1).

The rats' hearts were harvested and fixed in 4% formaldehyde solution for at least 24 h and embedded in paraffin. The paraffin-embedded specimens were cut into sections (3 μm thick) and stained with hematoxylin-eosin. The images were digitally captured (magnification: ×400) using a Leica DM3000 microscope (Leica Biosystems, Germany). Cardiomyocyte stained cross-sectional areas of each group were measured and digitalized by Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA). CSA/control ratios of each group were calculated and analyzed [28 (link)].

Proximity ligation assay (PLA) was performed using the Duolink^®In Situ Detection Reagents Orange kit (#DUO92007, Sigma-Aldrich). Briefly, MPM primary cells (5x10⁴) were seeded onto round coverslips and allowed to grow to 70% confluence. Cells were fixed with 3% PFA, for 15 min at RT in the dark, and then permeabilized using 0.1% Triton X-100 for 10 min. Non-specific binding was blocked using Duolink Blocking Solution for 1h at RT. Cells were then incubated with primary antibodies diluted in Duolink Antibody Diluent O/N at 4°C. Following washing in Wash Buffer A (10 mM Tris, 150 mM NaCl, 0.05% Tween 20), cells were incubated with secondary antibodies conjugated with PLUS and MINUS probes, for 1h at 37°C. Negative controls included only single primary or secondary antibodies. After two washing steps in Wash Buffer A, cells were incubated with the ligation-ligase solution for 30 min at 37°C, followed by an incubation with amplification-polymerase solution, for over 90 min at 37°C. Cells were washed in the Wash Buffer B (200 mmol/L Tris, 100 mmol/L NaCl) and coverslips were mounted in Fluorescence Mounting Medium with DAPI. Images were acquired using Leica DM3000 microscope (Leica, Wetzlar, Germany) and a Leica DFC320 digital camera.

Histological sections (8 μm) were cut from paraffin‐embedded tissue, mounted on microscope glass slides, and dried overnight in an incubator at 37°C. Sections were stained with hematoxylin and eosin. Digital images were captured with the use of a Leica DFC320 digital camera (Leica, Rijswijk, the Netherlands) at 20× magnification (Leica DM3000 microscope, Leica, Rijswijk, the Netherlands). Computerized morphometric analysis (Leica QWin V3, Cambridge, UK) of individual adipocytes was performed by measuring approximately 400 adipocytes per sample.

DECs, labelled with the fluorescent dye FAST DiI (Molecular Probes, Invitrogen), were seeded onto 8-chamber culture slides (BD Biosciences Discovery Labware, Milan, Italy), previously coated with 2 µg/cm² fibronectin (Roche). After fixation with 1% paraformaldehyde and permeabilization with FIX & PERM kit, solution B (Società Italiana Chimici, Rome, Italy), cells were incubated with 10 µg/mL primary monoclonal antibody (cloneF8/86) mouse anti-human vWF (Dako) or mouse anti-human vascular endothelial (VE)-cadherin (kindly provided by Prof. Dejana from the Institute of Molecular Oncology, Milan, Italy) for 1 h at room temperature (RT), followed by incubation with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Dako) for 1h at RT. Images were acquired with a Leica DM3000 microscope (Leica, Wetzlar, Germany) and collected using a Leica DFC320 digital camera (Leica).