The sections were incubated in a 3, 3-diaminobenzidine solution, counterstained with hematoxylin, dehydrated

in ethanol, Selleckchem TGFbeta inhibitor cleared in xylene, and coverslipped. Negative controls were treated in all assays (with the omission of primary antibodies). The sections were visualized using microscopic observation. Evaluation of the immunohistochemical findings IHC staining was assessed by two independent pathologists without knowledge of the clinical and pathologic features of the cases. A negative control array was concurrently undertaken showing < 1% nuclear staining in all specimens. All specimens were evaluated according to the 0–4 grading criteria (based on the percentage of 5-hmC-positive cells) and 0–3 grading criteria (based on the staining intensity) [11]. The 5-hmC score was calculated as the score of the cell count × the score of intensity. The median 5-hmC score was used as a cut-off in subsequent analyses. For IDH2 quantification, BI 2536 nmr photographs of three representative fields were captured under high-power magnification (200×) using Leica Qwin Plus v3 software; identical settings were used for each photograph. The 5-hmC and IDH2 density were counted using Image-Pro Plus v6.2 software (Media Cybernetics Inc., Bethesda, MD). The

integrated optical density of the CB-839 price area positively stained for IDH2 in each photograph was calculated, and its ratio to the total area of each photograph was considered to be the IDH2 density. The median IDH2 density was used as a cut-off in subsequent analyses. Statistical analysis The data were analyzed with SPSS 19.0 software, as previously described [23]. A P value <0.05 was considered statistically significant. Results Immunohistochemical features in TMA Using hematoxylin and eosin staining, the cancer cells were found to be relatively homogenous within a tumor (excluding necrotic,

DNA ligase hemorrhagic, and fibrotic components). Representative cases of immunohistochemical staining are shown in Figure 1. We observed 5-hmC staining primarily on the nuclei of the tumor cells and hepatocytes; IDH2 staining was observed primarily in the cytoplasm of the HCC cells. Most of the stromal cells were negatively stained, although sporadic positive staining of these cells was observed. Figure 1 Expression of 5-hmC and IDH2 in HCC samples (training cohort, n = 318). Representative HCC tumor samples show the expression of 5-hmC (brown in the nucleus of HCC cells) and IDH2 (brown in the cytoplasm of HCC cells). Scale bar, 200×, 200 μm. Correlations of 5-hmC and IDH2 expression with clinicopathologic characteristics The correlations of 5-hmC and IDH2 expression with the clinicopathologic characteristics are shown in Table 1 and Additional file 2: Table S2. In the training cohort, 5-hmC expression correlated with sex (P =0.007) and AFP (P <0.001). IDH2 expression only correlated with tumor differentiation (P =0.017) (Table 1).