Hepatic Steatosis Analysis.

Livers were weighed whole before the right lateral lobe was excised from the whole liver and fixed in 10% neutral buffered formalin overnight at 4 °C and then soaked in 30% sucrose (vol/vol) for 48 h; 1-cm × 1-cm segments were excised from the lobe and embedded in OCT compound (Tissue-Tek) before 10-µm sections were cryosectioned and air-dried onto slides. Slides were stained with oil red O in 60% isopropanol to visualize lipid deposition, as previously described (52), before histomorphometric analysis. ImageJ version 1.48 software (NIH) was used to quantify oil red O-positive staining. Three separate images were analyzed from three distinct sections taken at least 200 µm apart.