Histopathological Examination
Collected tissues were fixed and sectioned using a standard protocol developed by the Veterinary Diagnostic Laboratory at Washington University (15), ensuring each animal was examined for the same organ slice. After sectioning, the samples underwent processing with isopropyl alcohol, xylene, and paraffin using a tissue processor. Once placed in the histo-cassettes, the material was embedded in paraffin forming blocks for histological sectioning. Histological sections of approximately, 3μm were stained with hematoxylin and eosin to determine the general morphological pattern and cellular infiltration in the heart, kidneys, and liver. Also, Masson’s trichrome staining to identify collagen fibers, staining blue, and connective tissue in the heart and liver. To detect mucopolysaccharide accumulation in the mesangium and basal membranes of the glomeruli and tubules of the kidneys and for assessing glycogen accumulation in hepatocytes, tissue were stained with periodic acid-Schiff (PAS). Sections were examined using light microscopy using a Zeiss AxioLab 4.0 microscope at magnifications of ×40, ×100, ×200, and ×400. AxioVision 7.2 software for Windows was used for image capturing.