TSP-1-null mice were kindly provided by Dr. Jack Lawler (Beth Israel Deaconess Medical Center, Boston, MA).12 Male wild-type (WT) and TSP-1-null mice, at 8-12 weeks old (C57BL/6 background), were used for the experiments. The two anterior lobes (i.e., median and left lateral lobes), which comprise

70% of liver weight, were resected, whereas the caudate and right lobes were left intact. This study was approved by the institutional animal care and use committee. For histological analyses, liver samples (the same lobe from each mouse) were either directly frozen in OCT compound (Tissue-Tek; Sakura Finetek, Trichostatin A in vitro Tokyo, Japan) or fixed overnight in 4% paraformaldehyde INCB024360 purchase in phosphate-buffered saline (pH 7.2) and dehydrated in a graded alcohol series and embedded in paraffin. Then, the materials were sectioned at a thickness of 5 μm. Immunofluorescence (IF) and immunohistochemical (IHC) staining was performed as described previously.13 The negative control staining was performed without the addition of primary antibody. Immunostained slides were viewed under

a Leica DM 5500B microscopic system (Leica Microsystems, Buffalo Grove, IL). A minimum of 10 different images were randomly selected, and the data shown are representative of the results observed. Western blotting analysis was performed as described previously.13 The same lobe from each mouse was used for protein isolation and subsequent analysis. ImageJ software (version 1.40) was used for densitometric analysis. Mice received an intraperitoneal injection of 5-bromo-2-deoxyuridine (BrdU; 100 mg/kg; Roche

Applied Science, Indianapolis, IN) 2 hours before sacrifice. Six random visual high-power fields (0.64 mm2 per field) per mouse were evaluated to determine the number of BrdU-positive nuclei in hepatocytes and nonparenchymal cells. Nonparenchymal cells were defined as cells with smaller, irregularly learn more shaped nuclei, compared with larger, circular nuclei of hepatocytes, as previously described.14 All BrdU-positive cells, from both cell types, were summed at each time point. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis was performed using an in situ apoptosis detection kit (Roche). Six visual high-power fields (0.64 mm2 per field) per mouse were evaluated to determine the number of TUNEL-positive nuclei. The antibodies used for analyses are summarized in Supporting Table 1.