All experimental procedures had been accredited through the Institute of Biosciences and Engineering Institutional Animal Care and Use mittee with protocols 08058 entitled The Affect of Obesity and Weight problems Treatment method in Cancer and 10022 entitled BetaK lotho FGFR while in the liver. Tumor and tissue sample harvesting Mice had been sacrificed, breast tumors and breasts were re moved, along with a plete autopsy for all breasts and tu mors in each and every mouse was carried out. The samples had been evaluated by a pathologist in the blinded manner over the basis of H&E stained sections, and the breast alterations have been classified as hyperplasia, adenoma and duct carcin oma. The autopsy samples of lymph node, lung, liver and brain have been evaluated histopathologically for the presence of metastases. Tissue processing and immunohistochemistry Breasts and breast tumors have been removed from mice and fixed in 4% paraformaldehyde PBS for 4 h.
Fixed tissues had been processed for ethanol dehydration and par affin embedding as described Paraffin embedded tissue blocks have been serially sectioned and slide mounted. The sections have been deparaffinized and rehydrated before staining with H&E reagents, or primary and sec ondary antibodies for immunohistochemistry as indicated Ki67 and 5 bromo 2′ deoxyuridine selelck kinase inhibitor staining for mitotic index had been done according to the manufacturer’s protocol The section was then counterstained with hematoxylin and mounted with 1,1′ bis pyridiniudibromide media. The slides were analyzed by a pathologist and photographed digitally by light microscopy. Breast tumor incidence The tumor incidence within the age matched KO Tg and Tg mice was defined as the percentage of mice with tumor.
The palpable breast tumor foci found in every mouse in both groups were further confirmed by pathological examination on tissue sections under micro scope, and the incidence rate of breast tumor was calculated as percentage of the whole population in each and every group monthly Tumor multiplicity defined as number of breasts having tumor per PI-103 solubility mouse was also recorded monthly for the duration of the experiments. Tumor size was mea sured with a gauge. Mice have been sacrificed if the tumor load was excessive. Mouse survival analyses The life span of mice was the duration between the date of birth and the date of death or mandatory sacrifice due to tumor burden or illness. The curves for the rates of overall and breast tumor specific survival in both the FGFR4 deficient and WT mice with TGFa overexpression have been constructed using the Kaplan Meier method with the log rank test. Overall survival was expressed as the number of months to the date of death or sacrifice in both groups suffering from illnesses as a result of TGFa overexpression and or FGFR4 deficit.