"Twenty-four hours after the last treatment, mice were euthanized by overexposure to carbon dioxide and blood and liver tissue was obtained. The liver was weighed and cut into sections and fixed in 10% phosphate buffered formalin (PBF) for histological analysis or snap frozen in liquid nitrogen for subsequent RNA isolation and analysis for PFBA concentration.

Histopathology. For histological analysis, liver sections were fixed in 10% PBF for 24 h and then transferred to containers with 70% ethanol. Fixed liver sections were embedded in paraffin and 3-lm sections obtained. Representative sections from each liver were stained with hematoxylin and eosin (H&E) and examined by a pathologist using light microscopy. Hepatocyte hypertrophy and focal necrosis with inflammatory cell infiltration were scored based on incidence and severity with the following scoring system: grade 0 (normal); grade 1 (minimal); grade 2 (mild); grade 3 (moderate); and grade 4 (severe). Representative slides were used for immunohistochemical analysis of BrdU labeling to measure replicative DNA synthesis. BrdU staining was performed using the manufacturer’s recommended procedures (Calbiochem, San Diego, CA). Five frames from representative slide from each group were scored for BrdU labeling at 4003 magnification and are presented as the percentage of BrdU-positive cells.

Serum alanine aminotransferase assessment. Serum was obtained from mice and the concentration of l-alanine: 2-oxoglutarate aminotransferase (ALT) was measured using INFINITY125 ALT Reagent (Thermo Electron, Melbourne, Australia) according to the manufacturer’s recommended procedure."

Male PPAR-α Null Mice