17 Due to limited numbers of study subjects in each category, grades (G) and stages (S) were combined for the analyses as follows: steatosis grade, G0-1, G2, and G3 or G0-1 and G2-3; lobular inflammation grade, G0-1 and G2 (no case had G3); hepatocyte ballooning grade, G0 and G1-2; portal inflammation, G0, G1, and G2 or G0-1 and G2; and fibrosis stage, S0, S1-2, and S3-4 or S0-2 and S3-4. An overall diagnosis was also recorded using

the following diagnostic categories: steatosis (NAFLD but see more not NASH), suspicious for steatohepatitis (SH) adult pattern, which is also referred to zone 3 or Type 1 pattern, suspicious for SH pediatric pattern, which is also referred to zone 1 or Type 2 pattern, definite SH adult pattern, and definite SH pediatric

pattern. Initially, 30 of the 56 slides were stained for SHh (n = 20), vimentin (Vim, n = 6), or alpha-smooth muscle actin (α-SMA, n = 4), and the remaining slides check details were costained for Gli2 and keratin 7 (Gli2/K7, n = 26). To buttress sample size, two of the Gli2/K7 costained slides were additionally stained for Vim, two of the Gli2/K7 costained slides were additionally stained for α-SMA, and five of the SHh-stained slides were additionally stained for K7. Thus, in total 20 slides were evaluated for SHh, 26 for Gli2, 31 for K7, 8 for Vim, and 6 for α-SMA. This approach was necessary because the number of available slides was limited. In order to determine which slides were used for which stain, cases were grouped

according to histologic severity and then slides were randomly selected from each group for 17-DMAG (Alvespimycin) HCl immunohistochemistry (Ihc) in order to have roughly equivalent numbers of cases within each category of histologic severity. Details of the Ihc methods and antibodies have been published.18, 19 Positive staining for SHh, Vim, and αSMA was semiquantified as a percentage of the total surface area in low-power fields (×100 magnification) and graded into five ranked categories: G1 (less than 20%), G2 (20-39%), G3 (40-59%), G4 (60-79%), and G5 (≥80%). Nuclear positivity for Gli2 and cellular positivity for K7 were counted in five randomly selected high-power fields (HPFs, ×200 magnification). The average counts of K7+, Gli2+, K7+/Gli2−, and K7+/Gli2+ cells per HPF were calculated and used for the analyses. Furthermore, the intensity and histologic location of SHh and Gli2/K7 staining was evaluated by a hepatopathologist (C.G.). After excluding fragmented samples, 16 SHh slides, 18 Gli2 slides, and 25 K7 slides were used in this subanalysis.