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.