The sections were deparaffinized, rehydrated, and incubated with pepsin for 25 min at 37°C. The hybridization liquid that contains the Digoxigenin-labelled GW-572016 clinical trial RNA probes was placed on the sections, and the sections were then covered by parafilm and incubated at 42°C for 24 h in a moisture chamber. After hybridization, the slides were washed with different concentrations of SSC to remove the excess probe. The washed slides were incubated with diluted anti-Digoxigenin antibody conjugated HRP at 37°C for 2 h at room temperature, and colored with DAB (Zhongshan Jinqiao biotech company, Beijing, China) at 37°C for 30 min

with no exposure to light. The negative control samples PF-3084014 in vivo included the following: (i) RNase treatment (20 mg/ml) hybridization and (ii) use of neither probes nor anti-Digoxigenin antibody; the controls exhibited no positive signals. The positive controls included the positive slices provided by the kit and the combined use of ISH and IHC. The mRNA expression levels of Hsp90-beta and annexin A1 were Vorinostat independently evaluated by two pathologists (Wang JS and Li J). The mRNA levels of Hsp90-beta and annexin A1 exhibited positive staining in the cytoplasm. A specific scoring method for ISH was performed according to a previously published report [12]. The scoring method was as follows: according to the signal intensity, the signals

were divided into 4 groups, namely, absent (0), low (+), moderate (++), and

high (+++). For statistical analysis, we grouped the patients as low (0, +), moderate (++), and high (+++). Western blot The harvested cells were washed once with PBS, lysed with 2× sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample buffer (20 mM Tris, pH 8.0, 2% SDS, 2 mM dithiothreitol, 1 mM Na3VO4, 2 mM EDTA, and 20% glycerol), and boiled for 5 min. The protein concentration of each sample was determined using a Micro-BCA protein assay. In all samples, 30 μg of the total cellular protein was loaded on a 10% SDS-PAGE gel and electrophoretically separated. The proteins were transferred Phloretin to polyvinylidene difluoride membranes. The membranes were blocked for 2 h at 37°C in 20 mM Tris, pH 8.0, 150 mM NaCl, and 0.05% Tween 20 (TBST) containing either 5% BSA or 5% nonfat dried milk. The membranes were incubated with various antibodies (for immunoblotting with anti-Hsp90-beta 1:200 and annexin A1 antibody 1:400) overnight at 4°C. The primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies, and after three washes with TBST, positive signals were visualized using the enhanced chemiluminescence method. All experiments were performed for three separate times. Statistical analysis The associations between the expression status and clinico-pathological parameters were analyzed using the χ 2, Fisher’s exact, and McNemar tests.