For histological review of hematoxylin and eosin (H&E)–stained liver sections by light microscopy (Eclipse Meta Morph V 5.0.7, Nikon, West Lafayette, IN), the liver was diced into 5 mm×5 mm sections, fixed in 4% paraformaldehyde for 48 hours, and then embedded in paraffin. Tissue sections (4 µm) were prepared using a microtome (Reichert Scientific Instruments, Buffalo, NY) and placed on glass slides. H&E staining was performed according to standard techniques. For immunohistochemistry, paraformaldehyde-fixed paraffin-embedded liver tissue sections were deparaffinized, hydrated and incubated with antibodies against Mac-2 (1∶250, eBioscience, San Diego, CA) and sonic hedgehog (Shh, 1∶500, Santa Cruz Biotechnology, Santa Cruz, CA). Bound antibodies were detected using Vectastain ABC kit and diaminobenzidine as a substrate (both Vector Laboratories, Burlingame, CA) and the tissue slices were counterstained with methyl green or hematoxylin. To quantify Mac-2 immunohistochemical staining, ten random pictures of 20× fields per section were assessed by morphometry (KS 400 software, Carl Zeiss). Liver fibrosis was quantified using Sirius red staining. Direct red 80 and Fast-green FCF (color index 42053) were obtained from Sigma-Aldrich Diagnostics. Liver sections were stained, and red-stained collagen fibers were quantified by digital image analysis as previously described by us in detail . The fluorescent terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay on liver tissue (In situ cell death detection kit, Roche, Indianapolis, IN) was performed on frozen liver sections. Briefly, liver tissue samples were cryopreserved in Tissue Tek OTC compound (Takeda, Deerfield, IL) immediately after removal. Tissue sections were cut at 5 µm on a cryomicrotome (Leica, Buffalo Grove, IL), air dried and stored at –80°C until use. The TUNEL assay was then performed using the manufacturer's protocol and tissue slices were mounted with ProLong Gold antifade reagent with DAPI (Life Technologies, Grand Island, NY). Apoptotic cells were quantified by counting TUNEL-positive nuclei in 20 random microscopic fields (20×) using a fluorescent microscope (Eclipse 80i, Nikon, West Lafayette, IN). Full paper
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