*4.5. Hematoxylin and Eosin (H&E) Staining*

Histological changes in the liver tissue were analyzed by H&E staining (Sigma Aldrich., St Louis, MO, USA). Mice were perfused with a fixative solution containing 4% paraformaldehyde solution, and the liver was isolated and incubated in the same fixative solution overnight at 4 ◦C. The liver tissues were embedded in paraffin after washing three times. The paraffin blocks were sectioned to a thickness of 5 μm and air-dried on gelatin-coated slides. For H&E staining, the paraffin was removed from the liver tissue sections with xylene, and the tissue sections were rehydrated with graded alcohol series (100% to 70% EtOH). The liver tissue section was washed with tap water for 5 min, and the section slide was immersed in hematoxylin solution for 5 min. After checking the degree of hematoxylin staining, eosin staining was performed for 1 min. The sections were dehydrated through a graded series of EtOH (70% to 100% EtOH, each 3 min), removed from xylene, and mounted with mounting medium (Fisher Chemical, Geel, Belgium). The stained part was photographed using a BX61VS microscope (Olympus, Tokyo, Japan).

#### *4.6. TUNEL Staining*

The apoptotic signal in the testes was assessed using the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI, USA) according to the manufacturer's protocol. The TUNEL staining was carried out as described previously [56]. Deparaffinized liver tissue sections were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature, washed three times in PBS, and permeabilized with 20 μg/mL proteinase K solution for 10 min at room temperature. After three washes in PBS, the slides were refixed in 4% paraformaldehyde for 5 min at room temperature. The slides were washed in PBS for 5 min and equilibrated in an equilibration buffer for 10 min. The liver tissues on the slides were labeled with a TdT reaction mix for 60 min at 37 ◦C in a dark, humidified chamber. The reaction was stopped with a 2 × SSC solution, followed by washing three times in PBS. Counterstaining was carried out by incubating with 5 μg/mL PI for 10 min at room temperature in the dark. TUNEL-positive cells were observed using a confocal laser scanning microscope (Olympus).
