Five years follow-up was performed, and all patients had complete follow-up until death. Overall survival time was Entinostat 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 Streptavidin Biotin 2 System (BioGenex; San Ramon, CA, USA). Following BAY 80-6946 in vitro 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 Selleck GF120918 washed in PBS and color-developed by diaminobenzidine solution (Dako Corporation, Carpinteria, 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 immunohistochemical staining. Assessment of immunohistochemical staining was Casein kinase 1 evaluated 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 number 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 (moderate) 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 further 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 quantitative RT-PCR analysis according to the conventional protocols of Tangdu hospital [18].