The tissues were fixed in 10% buffered formaldehyde solution for pathological diagnosis and immunohistochemical staining. Histopathological diagnosis of each neoplastic tissue was performed according to the World Health Organization criteria by the Department of Pathology, Pusan National University Hospital. Clinicopathological staging was determined by the TNM classification. All patients www.selleckchem.com/products/VX-770.html had gastric cancer that was confirmed histologically, and tumour samples were checked to ensure that tumour tissue was present in more than 80% of the specimens. Follow-up data were collected until December 2008 or until the patient’s death, and the occurrence of metastasis and/or local recurrence was recorded.

Immunohistochemistry Immunohistochemical staining with rabbit anti-human stathmin1 polyclonal antibody (Cell Signaling Technology, Danvers, MA, USA, 1:50) and with rabbit anti-Ki-67 polyclonal antibody (Novocastra, Newcastle, UK, 1:200) was performed on 4-��m sections of paraffin-embedded specimens. Briefly, after deparaffination and hydration, the slides were incubated in 0.3% H2O2 for 30min to block endogenous peroxidase, after which the sections were blocked for 1h at room temperature with 10% blocking serum in phosphate-buffered saline (PBS) before reacting overnight with anti-stathmin1 antibody at 4��C in a moist chamber. For Ki-67 immunostaining, antigen retrieval was performed (boiling for 20min) in 10mM citrate buffer (pH 6.0). After incubation with the primary antibody, the specimens were washed three times in PBS and treated with secondary antibody at room temperature for 2h.

After washing three times in PBS, the specimens were treated with ABC reagent (Dako, Carpinteria, CA, USA), followed by colour development in 3,3��-diaminobenzidine tetrahydrochloride (Dako). Finally, the slides were lightly counterstained with haematoxylin, dehydrated with ethanol, cleaned with xylene and mounted. As a negative control, duplicate sections were immunostained without exposure to primary antibodies. To quantify the stathmin1 protein expression, the mean percentage of positive tumour cells was determined by scoring at least 1000 tumour cells of at least five random fields at �� 400 magnification in each section. The intensity of tumour cell staining was determined relative to that observed in adjacent endothelial cells (0, 1, 2 and 3 for negative, weak, moderate and strong). The percentage of positive tumour cells and staining intensity were then multiplied to produce a stathmin1 immunohistochemical staining Anacetrapib score. Cases with a stathmin1 score greater than 10 were defined as positive. For Ki-67 immunohistochemistry, the number of positive tumour cells was counted in 10 representative visual fields of each xenograft.