62 Ma et al. tive enough to differentiate between benign tumors and malignant tumors [20], we also performed smooth muscle actin (SMA) and PCNA immunohistochemistry staining to identify the differences among different histological types of rat mammary tumors. In addition, all tumors were eval- uated for estrogen receptor α (ERα) by immunohistochem- istry. II. Material and Methods Animal experimental procedure and care of laboratory animals followed the Guidelines for Animal Experiments of Peking University. After one week of acclimation to com- mercial powder chow and water, all of the 48 6-week old female Sprague-Dawley (SD) rats (Beijing Vital River Laboratory Animal Company, Beijing, China) received one intragastric intubation of 5 mg DMBA (Sigma-Aldrich, St. Louis, MO) dissolved in 0.5 ml corn oil. Twenty-four hours after DMBA administration, rats were assigned at random into 3 groups of 16 animals each and given 1 of 3 test solutions: milk, estrone sulfate solution or tap water for 20 weeks. The milk was purchased everyday at local super- market. Estrone sulfate (Sigma-Aldrich) was dissolved in tap water at a concentration of 100 ng/ml. Food intake and body weight were recorded weekly. Rats were palpated weekly to monitor tumor development. During the 20th week after DMBA administration, complete autopsies were performed. All organs were examined for gross abnormalities. Visible mammary tumors were rapidly excised and weighed. To carry out histological examination, the tumor with the adjacent normal gland was fixed in 10% neutral buffered formalin and embedded in paraffin. Tissue sections were cut at 2 µ m and stained with HE. For immunohistochemistry analysis, the following antibodies were used: mouse anti-alpha SMA monoclonal antibody (DAKO Corp., Carpinteria, CA, USA), mouse anti-PCNA antibody (clone, PC10; DAKO Corp.), or mouse anti-human ERα antibody (clone, ID5; DAKO Corp.). Anti- mouse and anti-rabbit, labeled polymer, horseradish peroxi- dase (DAKO Corp.) were applied to the sections as a second antibody. For ERα staining, paraffin sections were cut one day prior to immunohistochemistry staining because the immu- noreactivity of ERα in paraffin sections can diminish over time [9]. Procedures of immunohistochemistry staining for SMA, PCNA, and ERα have been described elsewhere [9, 12, 15]. Briefly, after removing paraffin wax in three changes of xylene and dehydration in a series of graded ethanol, sections were autoclaved at 121°C for 10 min in citrate buffer (pH 6.0) for antigen retrieval. Endogenous peroxidase was blocked by immersing the sections in 3% H2O2 for 10 min. Sections were incubated for 1 hr at room temperature with the primary antibody. Tissue sections were then reacted with the conjugated second-antibody, horse- radish peroxidase, for 40 min at room temperature. Visual- ization of SMA, PCNA, and ERα was performed with diaminobenzidine for 3 min. After counterstaining with haematoxylin for 10 s, sections were dehydrated in a series of graded ethanol, cleared in 3 changes of xylene and coverslipped for light microscopic examination. We performed histological diagnosis according to the classification described by Russo and Russo [21]. Statistical analysis Fisher’s exact probability test was used to compare the percentage of animals with tumors and tumor histology type in each group. Mean number of tumors per rat and PCNA labeling index among the three histological types were analyzed by one-way ANOVA followed by LSD multiple comparison tests. Statistical significance was set at p<0.05. Data analyses were performed using SPSS version 11.0 for Windows (SPSS Inc., Tokyo, Japan). III. Results Prevalence of mammary tumors During the period of observation, 48 rats developed 103 mammary tumors. Multiple tumors of different sizes were frequently found in the same rat. Results of mammary tumor development are shown in Table 1. The first tumors were detected at week 6 after DMBA administration in the milk and estrone sulfate groups. Palpable tumors in the water group did not appear until week 9 after DMBA ad- ministration. At week 20 after DMBA administration, tumor incidence was 75% (12/16) in milk group, 69% (11/16) in estrone sulfate group, and 38% (6/16) in water group. Milk group showed a significantly greater incidence in tumor development compared with the water group (p=0.037). Tumor incidence of estrone sulfate group was also high but not significantly different from that of the water group. Tumor incidence of milk group is comparable to that of estrone sulfate group. More than one third as many tumors were seen in the milk group as in the water group, and rats in the estrone sulfate group developed al- most twice as many tumors as did rats in the water group. No significant difference in number was observed between the Group I II III 16 16 16 I, milk group; II, estrone sulfate group; III, water group; SE, standard error. Table 1. Prevalence of DMBA-induced mammary tumors in three groups No. of rats Tumor incidence of rats (%) Total number of tumors Mean tumor No./rat (mean±SE) 75 69 38 53 33 17 3.3±0.75 2.1±0.51 1.0±0.42 p p=0.009