Semi automated microtome

Following animal scarification, the whole hearts were removed and cut into two slices, then cleaned from blood and epicardial fat tissue. Thereafter, heart slices were immobilized in plastic tissue cassettes and fixed in 10% neutral buffered formalin for no less than 48 hr. Heart tissues were processed into dehydration using four series changes of ascending concentration of ethanol alcohol (60%, 70%, 90%, and 100%). Afterward, tissue samples were cleaned with xylene and embedded in paraffin; the tissue-wax blocks were trimmed and sectioned to 5 µm using a semi-automated microtome (Leica-Germany). Furthermore, tissue sections were mounted and fixed on glass slides using a hot water bath and dried with a hot plate for 30 minutes. Later on, heart sections were deparaffinized by dipping into two changes of xylene each for 5 minutes and then rehydrated with four descending concentrations of ethanol (100%, 90%, 70%, and 60%) each for 5 minutes as well. Finally, tissue sections were stained with Harris’s hematoxylin and eosin (H&E) technique, then as last step sections were cleaned with xylene, dried, and covered by glassy cover-slips using mounting medium Distrene-Plasticizer Xylene (DPX).

Liver tissue samples collected at necropsy were processed for histopathology using an automated tissue processor (Leica, Germany) and paraffinized tissue blocks were prepared. Thin paraffin sections were cut at 4μm thickness using a semi-automated microtome (Leica, Germany). Tissue sections were then stained with Hematoxylin and eosin (H&E) and were examined under Olympus microscope BX57 and images were captured at 10× and 40× magnifications using DP71 digital camera system.
Histopathology was evaluated both qualitatively and quantitatively under the supervision of a qualified veterinary pathologist. The extents of vacuolar degeneration and necrosis were assessed by semi quantitative scoring criteria (Grade 0; for absence or less than 5% area affected, Grade 1; mild/minimal or less than 25% area affected, Grade 2; moderate or about 50% area affected and Grade 3; severe or more than 50% area affected). In addition, the number of mitotic figures was counted in the entire histology section and was expressed as number per unit area.