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Copyright by Eunyoung Park 2007 - The University of Texas at Austin

Copyright by Eunyoung Park 2007 - The University of Texas at Austin

slides showed

slides showed negative staining, four more random slides were selected and stained with CK20 to confirm negative staining. MMP-2 (AB19167, Chemicon, Temecula, CA), MMP-9 (AB16306-50, Abcam, Cambridge, MA), total Akt (#4691, Cell Signaling, Beverly, MA), and phospho-Akt (Ser 473) (sc-7985-R, Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were also used for immunohistochemistry. Three consecutive 4 μm sections were stained with CK20, MMP-2, and MMP-9 or CK20, total Akt, and phospho-Akt in this order. Tissue sections were deparaffinized in xylene, and rehydrated in descending ethanol series to water. Next, endogenous peroxidase was blocked by incubating with 3% hydrogen peroxide for 10 min. Antigen retrieval was performed by heating tissue sections in 10 mM citrate buffer (pH 6.0) for 10 min in a microwave followed by a 20 min cool down. Non-specific antibody binding was blocked with Biocare Blocking Reagent (BS966M, Biocare, Phoenix, AZ) for 10 min. A 1:100 dilution of CK20, a 1:100 dilution of MMP- 2, a 1:500 dilution of MMP-9, a 1:100 dilution of total Akt and a 1:50 dilution of phospho-Akt were used for primary antibody incubation. The samples were then incubated with horseradish peroxidase conjugated anti-rabbit (#RMR622, Biocare, Concord, CA) or anti-goat (#GHP516L, Biocare) secondary antibodies. The slides were the counterstained, mounted and observed by light microscopy. All immunohistochemistry was performed by the Histology & Tissue Processing Facility Core in the University of Texas M.D. Anderson Cancer Center. 103

Statistical Analysis Values shown are the mean ± SEM. Statistical tests other than tumor incidence were performed using SPSS (Apache Software Foundation, Wilmington, DE, version 12.0 for Windows). Fisher’s exact test was used for the statistical analysis for tumor incidence. Body weight, food intake, and tumor multiplicity were analyzed using two- tailed t-tests comparing each vitamin A concentration to control (2,400 IU/kg). Results were considered significantly different at P < 0.05. RESULTS Food intake and Body Weight Analysis At the beginning of the chemotherapy study, each group of mice (n=15 per group) had a mean body weight of 17.7 ± 0.02 g. Five weeks after tumor injection, the average body weight of the group consuming the diet containing 200,000 IU vitamin A/kg (17.7 ± 0.01 g) diet was less than that of the mice consuming the control diet (19.4 ± 0.12 g) (Fig. 5.1A). After consuming the diets for nine weeks, there were no significant differences in body weight between control mice, and those consuming 200,000 IU vitamin A/kg diet in the chemoprevention study. No mice in the chemoprevention study lost weight; both groups gained 4.8 ± 0.13 g of body weight. However, most weight gain was before tumor injection. There were no differences in food intake among the different dietary groups for both the chemotherapy and chemoprevention experiments. However, the amount of 104

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