Written informed consent was obtained from all patients and the study was approved by our institutional ethics committee. This investigation conformed to the principles outlined in the Declaration of Helsinki. Immunohistochemistry Paraffin-embedded sections (4 μm), Vistusertib molecular weight including tumor nests were obtained. Sections were deparaffinized and soaked in PBS prior to immunohistochemical analysis. Sections were also soaked in 3% H2O2 for 30 minutes in order to block endogenous tissue peroxidase, followed by treatment with

bovine serum for 30 minutes in order to reduce nonspecific binding. The DLL4 antibody (rabbit polyclonal; ab103469; Abcam) was diluted to 1:200, and incubated at room temperature for 12 hours. Sections were rinsed in PBS and visualized

by standard techniques for labeled avidin-biotin immunoperoxidase staining. Then, DLL4 was visualized using a DAB Substrate Kit. The slides were briefly counterstained with hematoxylin and mounted aqueously. Human brain tissue was used as a positive control for DLL4 expression (Figure 1). DLL4 positivity of four gastric cancer cell lines was examined by the same procedures of paraffin-embedded tissue without deparaffinization. Figure 1 DLL4 expression in brain tissue. DLL4 expression was identified in the brain tissue CYT387 clinical trial as a positive control of DLL4. Detection of DLL4 expression in gastric cancer cell lines by western blot analysis Gastric carcinoma cell lines, MKN28, MNK45, KATO-III, and NUGC4 were purchased from the Japanese Physical and Chemical Institute, Tokyo, Japan. Sitaxentan They were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C in a cell incubator. All cells were harvested by centrifugation, rinsed with phosphate buffered saline (PBS), and subjected to total protein extraction in an immunoprecipitation assay buffer lysis buffer. The separation of nuclear extract and cytoplasmic

fraction was performed by a Nuclear/Cytosol Fraction Kit (K266-25 BioVison, USA). Cytoplasmic and nucleus DLL4 expression were extracted to facilitate Western blot. Western blot analysis of DLL4 of gastric cancer cell lines was performed. Denatured proteins extracted from the nucleus and cytoplasm were separated on an SDS-polyacrylamide gel and transferred to Hybond membrane, which was then blocked overnight in 5% skimmed milk in TBS. For immunoblotting, the membrane was incubated for 15 minutes with mouse antibody against DLL4 (1:2000). Then, it was rinsed by TBST and incubated with anti-house IgG conjugated to horseradish peroxidase for 15 minutes. Bands were visualized with X-ray film (Fuji, Japan) by PRN1371 order ECL-Plus detection reagents. After that, the membrane was washed with WB Stripping Solution (Nakarai, Tokyo, Japan) for 15 minutes and treated as described above except anti-β-actin antibody (sc-47778, Santa Cruz, 1:1000) as the internal control.