Hematoxylin and eosin h e

Serial 3 μm sections were stained using Hematoxylin and Eosin (H&E) (Bio Optica, Milano, Italy), in order to highlight the degree of inflammation, and Masson’s Trichrome (Bio Optica, Milano, Italy), to evaluate the deposition of connective tissue and fibrosis. Furthermore, human samples were stained with Periodic acid-Schiff (PAS, Bio Optica, Milano, Italy) to assess changes in the number of goblet cells. The stained sections were then observed under an Olympus BX51 Light Microscope (Olympus Optical Co., Ltd., Tokyo, Japan). Quantification of markers in mouse sections was performed on three whole sections and normalized against the total section area, while for human samples, four fields were randomly analyzed and normalized against the number of nuclei. Two independent pathologists performed blind evaluations.

For histological analysis at light microscopy, samples were fixed with 4% paraformaldehyde overnight at 4 °C, embedded in paraffin, and serial-sectioned (5 μm) with a Leica Jung Multicut 2045 Microtome (Leica, Wien, Austria). After deparaffinization and rehydration through a graded ethanol scale, sections were stained with hematoxylin and eosin (H.E.) (Bio-Optica, Milan, Italy), for a general morphological view, with Masson Trichrome (M.T.) with aniline blue kit (Bio-Optica, Milan, Italy) to highlight the collagenic component of capsular connective tissue and silver impregnation (S.I.) histoenzymatic kit (Bio-Optica, Milan, Italy) to highlight argyrophilic neurofibrils in the capsular connective. Samples were observed with a Nikon Eclipse Ni light microscope (Nikon, Tokyo, Japan) and data were recorded with a DS-5M-L1 digital camera system (Nikon, Tokyo, Japan).

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.