Sucrose

Eight-week-old male CBA mice were purchased from animal facilities at Ru À der Boškovic ´Institute. Animals were maintained in standard conditions on chow diet or on a high-fat diet containing 58% fat, 16.4% proteins, and 25.6% carbohydrates (Mucedolla, Italy) for a period of 20 weeks. At the end of the experiment, the animals were euthanized by an overdose of Ketamidor 10% (Richter Pharma AG, Wels, Austria). The liver was immediately removed, fixed in Bouin's solution (picric acid, saturated aqueous solution-75 ml; formalin, 40% aqueous solution-25 ml; acetic acid, glacial-5 ml) for at least 4 h, washed in PBS, and preserved by immersion in 30% (w/v) sucrose (Kemika, Zagreb, Croatia) in PBS overnight. Small pieces of the liver tissue were immersed in an OCT compound (Sakura, Netherland), frozen in isopentane, cooled by liquid nitrogen, and cryosectioned at 8 μm in a freezing cryostat (Leica, Austria). After incubation in a series of tap and distilled water and ethanol, the frozen sections were incubated in a Sudan III working solution (0.1% Sudan III solution in 70% alcohol) for 30 min and then washed in distilled water. The sections were then counterstained with hematoxylin and viewed under a light microscope.