Fan et al. BMC Cancer 2011, 11:320 http://www.biomedcentral.com/1471-2407/11/320 expressed CD133 protein; moreover, the 5-year survival rate in the CD133-positive group was worse than that in the CD133-negative group [23]. On the contrary, in our study we detected the expression of CD133 in human CC specimens by immunohistochemistry and analyzed its correlation with clinical and histopathological fea- tures. We found that CD133 was expressed on apical membrane of normal bile ducts and apical membrane or/and in the cytoplasma of CC tissues. The positive expression of CD133 on tumor cells was significantly correlated with well or moderately differentiated CC cases and predicted a better prognosis for the patients. Methods Clinical samples Paraffin-embedded liver tissues from 54 consecutive patients with CC were retrieved from the pathology archives of Xijing Hospital between 2003 and 2007. In addition, 5 normal liver tissues were collected from the donor livers. The CC patients consisted of 28 males and 26 females with a mean age of 59.4 years (range from 34 to78 years). And the tumor specimens were com- posed of 25 cases of intrahepatic CC (ICC) and 29 cases of hilar CC (HCC) (Table 1). The patients’ charts were reviewed and those who had pre-operative radiation or chemotherapy, or were immunocompromised, were excluded. All the histological slides were re-examined by two pathologists to ensure correct diagnosis of CC and the degree of its differentiation was evaluated in each case. Histological grading of CC was assessed based on the World Health Organization Classification of Tumors [24]. Among the 54 cases, 21 were well-differentiated, 12 were moderately-differentiated and 21 were poorly- differentiated. For clinical staging, 18 cases were at stage I/II while the other 36 were at stage III/IV. Moreover, 23 patients were followed up for survival from the date of surgery to March 31, 2008, including only that cacer- specific death of CC, the details of which were listed in additional file 1. Specimen collection and study proce- dures were approved by the Ethics Committee of Xijing Hospital. Table 1 The origin and differentiation degree of the 54 cases of CC Origin Differentiation degree ICC HCC Subtotal Well-differentiated glandular carcinoma Moderately-differentiated glandular carcinoma Poorly glandular carcinoma Subtotal 7 5 13 25 14 7 8 29 ICC: Intrahepatic cholangiocarcinoma; HCC: Hilar cholangiocarcinoma 21 12 21 54 Page 2 of 8 Immunohistochemistry Paraffin sections of 4 μm thickness were deparaffinized and treated with 3% (v/v) hydrogen peroxide to block endogenous peroxidase activity. Heat-induced antigen retrieval was performed in 0.01 M sodium citrate (pH 6.0), followed by incubation in 10% (v/v) bovine serum albumin (BSA; Sigma, St Louis, MO, USA) in PBS (Phosphate Buffered Saline) at room temperature for 10 minutes to block the non-specific antibody-binding sites. The sections were then incubated overnight at 4°C with the rabbit monoclonal antibody against the human CD133 proteins (1:100 dilution; clone C24B9, Cell Sig- naling Technology, Danvers, MA, USA). Later on, a standard rapid EnVision technique (REAL™ EnVision™ Detection System, Peroxidase/DAB+, Rabbit/Mouse, Code K5007, Dako, Denmark) was used to detect the protein conjugates and develop the color. Finally, the sections were visualized after counterstaining with hematoxylin. Serial sections of CC were run in parallel with the primary antibody replaced by PBS and rabbit IgG1 (AMS/Immunokontact, Abingdon, UK) as blank and negative controls. The whole sections were screened for CD133 expression and the cholangiocarcinoma cases were defined as CD133 positive if CD133 staining was detected in more than 10% of the entire tumor area [25]. The slides were viewed under 100 × magnifications to define subcellular localization of the protein in the tumor glands. The evaluation of CD133 staining was carried out by two pathologists who had no knowledge of clinicopathological features of the cases. Statistics Statistical analyses were performed using Statistical Pro- gram for Social Sciences (SPSS) software (Version 17.0, SPSS Inc, Chicago, USA). A Spearman correlation was used to analyze the correlation between the expression of CD133 and the clinicopathological features. The Kaplan-Meier analysis was also used to estimate tumor- specific survival, and different groups were compared with the log-rank test. For multivariate analysis, the COX regression model was used [26]. All tests were two-sided. P < 0.05 was considered significant. Results CD133 Expresses on Normal Bile Duct Epithelia and Most of the CC Tumor Cells with Different Subcelluar Localization In the normal liver tissues, CD133 expression was found mainly on the apical membrane surface of bile ducts of all sizes (Figure 1A-C) but was not seen in hepatocytes, hepatic vessels and sinusoidal lining cells. In CC tissues, 40 of 54 cases (74%) were positive for CD133.