Histological Evaluation of Liver Injury and Fibrosis

Liver tissue from the same hepatic lobe of all mice was excised from EtOH, EtOH-P, and Con mice, fixed in 10% buffered formalin, and embedded in paraffin as described previously.^{32 (link)} Thin sections, obtained from paraffin blocks, were stained with hematoxylin–eosin for histological evaluation of liver injury and Masson's trichrome stain was performed for evaluation of liver fibrosis.^{33 (link)} Slides were examined, and images captured under bright field with a Leica DM5000 upright microscope (Leica Microsystems, Heidelberg, Germany). Liver sections were examined by two expert pathologists in a blind manner, using coded slides and not knowing their source. The mean of the results was used as data. Liver steatosis was classified using a semiquantitative scoring system divided into three fat grades (grade I=5–33% grade II=34–66% and grade III=67–100%).^{34 (link)}