The selleck chemical use of the human tissue in this study was approved by the Ethics Council of the Sun Yat-Sen University for Approval of Research Involving Human Subjects. Immunohistochemistry All 5μm thick paraffin sections were deparaffinized with xylene and rehydrated through graded alcohol washes, followed by antigen retrieval by heating sections in sodium citrate buffer (10 mmol/L, pH6.0) for 30 minutes. Endogenous peroxidase activity was blocked with 30 min incubation in 0.03% H2O2 in methanol. The slides were then blocked by incubation in normal goat serum (dilution 1:10) in PBS (pH 7.4) and subsequently GW-572016 cost incubated for monoclonal mouse IgG1 anti-Pim-1

antibody(sc-13513; Santa Cruz Biotechnology, Santa Cruz, CA, USA) with 1:30 dilution at 4°C overnight. Following this step, slides were treated with biotin-labeled anti-IgG and incubated with preformed avidin-biotin peroxidase complex. Control staining of the same sections was performed with the preimmune primary antibody, and no Pim-1 immunostaining was observed in these sections. The sections were briefly counter-stained with hematoxylin. IHC reactions for all samples were repeated at least three times, and

PF-3084014 mouse typical results were illustrated. Scoring and Statistical analyses The staining of Pim-1 was graded in each sample based on the intensity of the immunoreactivity in the cancer cells and was stratified as strong staining (3), moderate staining (2), weak staining (1) and negative (0). Using these criteria, the immunostaining results were evaluated independently by XPM and BH. The correlation of interobserver was calculated from the independent evaluations. For cases with discrepancy, a consensus was reached during a common

evaluation session. The statistical analyses were carried out by using SAS version 9.0 statistics software (SAS Institute, Inc., Cary, NC). Cell culture and lentiviral infection Bladder cancer Sirolimus concentration cell lines T24, UM-UC-3, 5637, J82 and RT-4 were purchased from the American Type Culture Collection. UM-UC-3 and T24 cells were grown in Dulbecco’s modified Eagle’s medium. 5637, J82 and RT-4 cells were maintained in RPMI 1640 with 10% fetal bovine serum and 1% (v/v) penicillin and streptomycin (100 μg/ml) and maintained at 37°C in a 5% CO2 atmosphere. The infection of lentivirus of Pim-1 siRNA was carried out as reported previously [15]. Western Blot Western blot was performed as described previously [16]. Briefly, the equal amounts of sample were resolved on a SDS polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Blots were incubated with the indicated primary antibodies overnight at 4°C and followed by detection with horseradish peroxidase-conjugated secondary antibody.