Nis elements d 3

Parts of the small intestine (duodenum, jejunum and ileum) and caecum fixed in 5% buffered formalin solution were embedded in paraffin using a standard procedure. Subsequently, an evenly spaced series of histologic section (50 μm intersection interval) were cut at 5 μm and stained with hematoxylin and eosin (H&E).
Histomorphometric analysis was performed on H&E-stained tissue sections using a Nikon Microphot FXA microscope equipped with a DS-Fi1 camera and the Imaging Software NIS Elements D.32 (Nikon instruments Europe B.V., Badhoevedorp, The Netherlands).
Villus height was measured from the tip to the crypt-villus junction and the crypt depth measured from the crypt-villus junction to the base as illustrated in Figure 1.

Myocardial tissue and epididymal fat pads were fixed in 4% paraformaldehyde for 24 h, routinely processed, and embedded in paraffin. Briefly, all tissue paraffin blocks were cut to a 5 µm thickness using a microtome. The paraffin sections (5 µm) were dewaxed and rehydrated using gradient alcohol and water. The sections were then washed with tap water and distilled water and then stained with hematoxylin and eosin (H&E) (Bio-Optica Milano SpA., Milano, Italy). For microscopic assessment, the images of heart sections were captured by a stereoscope (Nikon SMZ745T with NIS-elements D 3.2) with a 1× objective lens to observe the LV wall thickness, the cross-sectional area (CSA), the LV luminal area, and the wall-to-lumen ratio. These parameters were counted using Image J software (National Institutes of Health, Bethesda, MD, USA).
The measurement of the myocardium cell size and the area of cardiomyocytes was performed for 300 myocytes per group with a 40× objective lens using a Digital sight DS-2MV light microscope (Nikon, Tokyo, Japan). Mean values were obtained for 300 cells/group.
Epididymal fat sections were evaluated using a Digital sight DS-2MV light microscope (Nikon, Tokyo, Japan) with a 40× objective lens. Adipocytes were quantitated according to the cell size area (300 cells/group) using NIS-Elements software.

Biometric analyses were performed by measuring length (L, maximum axis of the oral disc), width (W, minimum axis of the oral disc), and height (h, oral–aboral axis) of each sampled polyp. Polyp body volume (V), calculated using the formula V = (L/2) * (W/2) * h * π, was used to estimate reproductive parameters (oocyte and spermary abundance, and gonadal index) as described in the next section. Polyps have been processed following the histological protocols as described in Goffredo et al. [18 , 19 ].
Cytometric analyses were carried out with an optical microscope using the image analyzer NIKON NIS-Elements D 3.2. The maximum and minimum diameters of the oocytes in nucleated sections and spermaries were measured. Spermaries were classified into five developmental stages and oocytes were classified as immature or mature, according to earlier studies on gametogenesis of this species [18 ].

All organ samples for histopathological examination were embedded in paraffin wax, sectioned at 4 µm and stained with Harris’ Haematoxylin and Eosin (H&E). The slides were viewed under light microscope (Nikon Eclipse 50i, Tokyo, Japan) installed with Nikon imaging software (NIS-Elements D 3.2, Tokyo, Japan). The histological changes were noted and scored as 0: none, 1: mild, 2: moderate and 3: severe. Specific lesions like necrosis, atrophy, haemorrhage, congestion, infiltration of inflammatory cells and hyperplasia were graded based on these criteria; 0: none, 1: 30% affected, 2: 30–60% affected and 3: > more than 60% affected (Xavier et al. 2010 ). All samples were duplicated and 5 microscopic fields of each slide were randomly selected for lesion scoring. The scores were reported as the average value of each lesion and average value for overall scoring.

Cytohistometric observations were performed with an optical microscope using the software NIKON NIS-Elements D 3.2. The maximum and minimum diameters of the oocytes in nucleated sections and spermaries were measured and classified into developmental stages according to earlier studies on gametogenesis in scleractinians [11 ,37 ,51 –54 ]. The presence of embryos in the gastrovascular cavity and mesenterial septa was recorded, and their stage of maturation identified [2 ,35 ]. The size of each reproductive element was determined as the mean of the two diameters [2 ,43 ].

Contractility of TGF-β1-treated and -non-treated cells was evaluated by a cell contraction assay in collagen I hydrogel. A 24-well plate was coated with 1 % BSA and incubated at 37 °C for at least 1 h. Cells from 3 donors were encapsulated in 1.81 mg/mL type I collagen with 4 technical replicates at a seeding density of 1.5 × 10⁵ cells/mL. Cells were cultured at 37 °C 2 % O₂ in αMEM with 10 % FBS. After 24 h, gels were photographed (Canon PowerShot SX50 HS) and well and gel diameters measured using NIS-Elements D 3.2 (NIKON Japan). Two gel diameters were averaged to calculate the gel area. The gel area after 24 h was divided by the well area, calculated from the well diameter.