Different concentrations of Genistein (0, 25, 50, 100, and 200 μM) was added to the cells to observe the effect of Genistein on VM. Animal model and CD34-PAS dual staining All animal experiments were
approved see more by the local animal ethics committee. Six week old female BALB/C nu/nu mice were purchased from Vital River Laboratory Animal Technology (Beijing, China). All experiments were performed in accordance with the official recommendations of the Chinese Community Guidelines. The xenografts were established using C918 cells , which were resuspended at a density of 1 × 107/ml. The suspension (0.1 ml/10 g body weight) was injected subcutaneously into the nude mice. After 6 days, tumor nodules were palpable. Then the mice were randomly assigned into control and Genistein groups: control (n = 5), injected PCI-32765 cell line intraperitoneally with 1% DMSO/day; Genistein (n = 5), injected intraperitoneally with Genistein 75 mg/kg/day. The treatment was continued every day for 30 days. At the end, mice were sacrificed by cervical decapitation and the tumors were removed and weighed. C918 xenograft specimens were fixed in 10% neutral buffered formalin and paraffin-embedded. Paraffin-embedded specimens were cut into serial
5-μm sections. And the sections were deparaffinized, rehydrated, and subjected to immunohistochemical and PAS double-staining. The immunohistochemistry was conducted with monoclonal mouse antibodies to the endothelium marker CD34 (1:50 dilution, Beijng, Zhong Shan Goldenbridge) to identify endothelium. DAB chromogen was used for the immunohistochemistry. CD34 staining helped to distinguish the PAS-positive network of VM from endothelium-lined micro vessels. Tissues were stained with PAS to identify the matrix-associated
vascular channels of uveal melanoma. Quantification of VM was performed as follow : The CD34-PAS dual staining sections were viewed at × 400. The channels defined as VM were lined by PAS-positive material Rutecarpine with red cells in the center of the channels, but not lined by CD34-positive endothelial cells. The mean VM count of ten areas was calculated as the VM density (VMD) respectively for each section. The mean VMD from 5 xenograft specimens in the Genistein and control groups were obtained as the final VMD count. Semiquantitative RT-PCR analysis The mRNA expression of VE-cadherin in C918 cells was analyzed by reverse transcription polymerase chain reaction (RT-PCR). At the end of Genistein treatment, total RNA from C918 and OCM-1A cells cultured on a type I collagen three-dimensional matrix was extracted using NSC 683864 chemical structure Trizol reagent (Invitrogen) as the manufacturer’s protocol. The first-strand cDNA was synthesized from 3 μg of RNA by standard reverse transcription (RT) methods, using M-MuLV reverse transcriptase (MBI Fermentas, Vilnius, Lithuania) and oligt (d) T primer according to the manufacturer’s instructions.