Histochemical Analysis of Liver Morphology and Immune Cells

Paraffin-sectioned livers (5 μm thickness) were stained with hematoxylin and eosin (H&E) to evaluate the morphological changes [40 (link)]. Another set of paraffin-embedded sections were immunostained as previously described [29 (link)], using rat anti-F4/80 antibody (1:100 dilution, MCA497, Bio-Rad), followed by biotinylated goat anti-rat IgG (1:200, Dako, Glostrup, Denmark) and streptavidin-horse radish peroxidase (HRP) conjugate (Zymed Laboratories, Inc., San Francisco, CA, USA). These slides were counterstained with Mayer’s hematoxylin. For the negative controls, normal rat IgG was used as primary antibodies. To stain for neutral lipids, frozen liver samples were cut into 10 μm thick sections using a Leica CM1850 UV Cryostat (Wetzlar, Germany). The frozen sections were stained with Oil Red O to visualize lipid droplets in the liver [40 (link)]. All the images were captured using an Olympus BX43 Upright Light Microscope (Olympus Corp., Tokyo, Japan) equipped with a Q-color 3 digital camera and analyzed using Image-Pro Plus 7.0 (Media Cybernetics, Rockville, MD, USA).