remove the peritoneal tumors for histologic and biochemical evaluation. Immunohistochemical analysis and microvessel density Six micrometer sections of formalin fixed and paraffin embedded tissue specimens were stained by an established method described previously. Sections were incu bated with antibodies specific for Factor VIII, vascular endothelial growth factor, cleaved caspase 3, human P450 vessel number high power field in sections. Three fields were counted per animal and the average was taken as the MVD of each tumor. Weatern blot analysis Cell lysates were prepared from tumor tis sues, electrophoresed through a 12. 5% SDS polyacrylamide gel, and blotted as described previously. The protein concentration was determined using Bradfords method.
The blots were probed with the following diluted anti bodies for 2 hr, cleaved caspase 3 at 1,1000 and B actin at 1,2000. The membranes were then incubated for 1 hr with the appropriate biotinylated secondary antibodies, these details trans ferred to avidin biotin peroxidase complex reagent, and incubated in this solution for 30 min. Diaminobenzidine was used as a substrate. aromatase, ER and FOXP1 at 4 C overnight. Slides were incubated with biotinylated species specific appropriate secondary antibodies for 30 minutes and exposed to avidin biotin peroxidase complex. Sec tions were treated with 0. 02% DAB as a chromogen and counterstained with hematoxylin. Microvessel density was determined as follows. The highly vascula rized areas of the tumor stained with an anti Factor VIII antibody were identified and Factor VIII positive micro vessels were counted within a high power field.
Single endothelial cells or clusters of endothelial cells, with or without lumen, were con Statistical analysis Survival rates were calculated by the Kaplan Meier method, and the statistical significance of differences in the cumulative survival curves selleck chemical Oxiracetam between the groups was evaluated using the log rank test. Other statistical ana lysis was carried out with the Student t test. A result was deemed significant at a P value 0. 05. Results Comparison of mRNA expression of ER in the ovarian cancer cell lines We determined mRNA abundance of ER in four ova rian cancer cell lines using real time quantitative PCR. We found that the level of ER mRNAin OVCAR 3 cells was significantly higher than that in other three cell lines.
Thus, OVCAR 3 was de fined as ER positive, whereas DISS, MCAS and TOV 112D were defined as ER negative. Evaluation of adverse effect caused by giving letrozole after ovariectomy Changes in the body weights of ovariectomized mice were evaluated. Body weights were 27. 9 1. 4 in mice given letrozole for 5 weeks and 28. 1 2. 4 in mice given vehicle, with no significant difference. All of the mice were healthy and did not exhibit self
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