Wang et al. Journal of Experimental & Clinical Cancer Research 2012, 31:44 http://www.jeccr.com/content/31/1/44 Page 3 of 7 WHO classifications [1] by two pathologists, with differ- ences resolved by careful discussion. A total of 76 males and 52 females (1.46:1) were enrolled in this study, and the median age was 42 years (range, 12–71). Thirty-two of the 128 gliomas were classified as low-grade [18 pilo- cytic astrocytomas (WHO I) and 14 diffuse astrocytomas (WHO II)], and 96 were classified as high-grade gliomas [38 anaplasia astrocytomas (WHO III), and 58 primary glioblastomas (WHO IV)]. None of the patients had received chemotherapy or radiotherapy prior to surgery. The clinicopathological features and the treatment strat- egies of all the patients were indicated in Table 1. Paraf- fin and snap-frozen sections of nonneoplastic brain tissues from 10 patients with intractable epilepsy were also included as controls. In addition, 20 glioma specimens [5 pilocytic astrocy- tomas (WHO I), 3 diffuse astrocytomas (WHO II), 3 anaplasia astrocytomas (WHO III), and 9 primary glio- blastomas (WHO IV)] were snap-frozen in liquid nitro- gen and stored at −80°C following surgery for real-time quantitative RT-PCR assay. Five years follow-up was performed, and all patients had complete follow-up until death. Overall survival time was calculated from the date of the initial surgical operation to death. Patients, who died of diseases not directly related to their gliomas or due to unexpected events, were excluded from this study. Immunohistochemistry assay Formalin-fixed, paraffin-embedded, sectioned tissues (4 μm thick) were immunostained using the Labelled Strep- tavidin Biotin 2 System (BioGenex; San Ramon, CA, USA). Following peroxidase blocking with 0.3% H2O2/ methanol for 30 min, specimens were blocked with phosphate-buffered saline (PBS) containing 5% normal horse serum (Vector Laboratories Inc., Burlingame, CA, USA). All incubations with mouse anti-human CLIC1 monoclonal antibody (1:175 dilution, Abcam,Cambridge, UK) were carried out overnight at 4°C. The specificity of this primary antibody has been demonstrated in previous studies of Wang et al. [11]. Then the specimens were briefly washed in PBS and incubated at room temperature with the anti-mouse antibody and avidin-biotin peroxidase (Vector Laboratories Inc., Burlingame, CA, USA). The specimens were then washed in PBS and color-developed by diaminobenzidine solution (Dako Corporation, Carpin- teria, CA, USA). After washing with water, specimens were counterstained with Meyer’s hematoxylin (Sigma Chemical Co., St Louis, MO, USA). Nonneoplastic brain tissues were used as control tissues and non-immune IgG was also used as negative control antibody for immunohis- tochemical staining. Assessment of immunohistochemical staining was eval- uated by two independent pathologists. The scores of the two pathologists were compared and any discrepant scores were trained through re-examining the stainings by both pathologists to achieve a consensus score. The num- ber of positive-staining cells showing immunoreactivity in cytoplasm for CLIC1 in ten representative microscopic fields was counted and the percentage of positive cells was calculated. The percentage scoring of immunoreactive tumor cells was as follows: 0 (0%), 1 (1–10%), 2 (11–50%) and 3 (>50%). The staining intensity was visually scored and stratified as follows: 0 (negative), 1 (weak), 2 (moder- ate) and 3 (strong). A final immunoreactivity scores (IRS) was obtained for each case by multiplying the percentage and the intensity score. Protein expression levels were fur- ther analyzed by classifying IRS values as low (based on a IRS value less than 5) and as high (based on a IRS value greater than 5). Real-time quantitative RT-PCR The mRNA expression of CLIC1 in glioma and non- neoplastic brain tissues was detected by real-time quan- titative RT-PCR analysis according to the conventional protocols of Tangdu hospital [18]. Especially, the primers were designed as follows: for human CLIC1, forward pri- mer, 5′- ATG GCT GAA GAA CAA CCG -3′, and re- verse primer, 5′- TTA TTT GAG GGA CTT TGA -3′; for human glyceraldehyde 3-phosphate dehydrogenase (GAPDH), forward primer, 5′- CCC ACT CCT CCA CCT TTG AC-3′, and reverse primer, 5′-ATG AGG TCC ACC ACC CTG TT-3′. Each sample was examined in triplicate and the amounts of the PCR products pro- duced were non-neoplasticized to GAPDH which served as internal control. Statistical analysis All computations were carried out using the software of SPSS version13.0 for Windows (SPSS Inc, IL, USA). Data were expressed as means±standard deviation (SD). The analysis of variance (ANOVA) was used to determine the statistical differences among the groups. A life table was calculated according to the Kaplan-Meier method. Hazard ratios for the time-to-event endpoint were esti- mated using the multivariate Cox regression analysis in a forward stepwise method to evaluate the effect of mul- tiple independent prognostic factors on survival out- come. Differences were considered statistically significant when p was less than 0.05. Results CLIC1 mRNA expression in human glioma tissues The expression levels of CLIC1 mRNA were detected in 20 glioma and 10 non-neoplastic brain tissues normalized to GAPDH. As shown in Figure 1A, the expression levels of CLIC1 mRNA were found to be distinctly increased in glioma tissues compared to non-neoplastic brain tissues,