Animals and carcinogen treatment
Copenhagen and Wistar-Furth rats (6-7 weeks old) were purchased from Harlan Sprague Dawley (Indianapolis, Indiana, USA), maintained on a 12h light/dark cycle, fed Harlan Teklad rat chow (6% fat; Harlan Teklad, Madison, Wisconsin, USA), and were given free access to water. After 1 week of acclimatization the rats were given an intraperitoneal injection of 50 mg/kg MNU dissolved in acidified normal saline.
Bromodeoxyuridine treatment and mammary whole-mountpreparation
At 20, 30, and 37 days after MNU treatment, five rats from each strain selected randomly were given an intraperitoneal injection of 50 mg/kg bromodeoxyuridine (Boehringer, Laval, Canada) dissolved in phosphate-buffered saline. Three hours later, they were killed and mammary whole-mounts prepared, using the technique we described previously [4].
Paraffin embedding, staining, and immunohistochemistry
Putative lesions in the whole-mounts were microdissected from the glands, cleared in xylenes, processed through three changes of paraffin wax, and then embedded in paraffin wax (Fisher, Whitby, Canada) for sectioning. Sections (4μ m thick) were placed on poly-L-lysine (Sigma, St Louis, Missouri, USA) coated slides and stained with hematoxylin and eosin. Positive identification of IDPs, DCIS, and adenocarcinomas was based on the criteria we used previously [4]. Serial sections from confirmed lesions were then used for cyclin D1, p16INK4a, and bromodeoxyuridine immunohistochemistry using established techniques [13,14,15]. Anti-cycin D1 and anti-p16INK4a antibodies were obtained from Santa Cruz (Santa Cruz, California, USA) and anti-bromodeoxyuridine antibodies from Boehringer. Archival rat mammary tumor tissue was used as a positive control for cyclin D1, because its overexpression has been reported in these tumors [8]. The levels of cyclin D1 in the stained samples were scored as negative (-), low (+), or high (++), based on number of positive cells in the lesion as well as staining intensity. Because the measurement of staining intensity was somewhat subjective, the coded samples were also scored independently by a second individual, with identical results. The percentage of cyclin D1-positive cells was determined as the number of positive cells divided by total cell number in a lesion.
The bromodeoxyuridine labeling index was determined by the number of bromodeoxyuridine-positive cells divided by total cells in a lesion. Small intestine from bromodeoxyuridine-treated rats or livers from partially hepatectomized rats were used as positive controls for staining.
For all immunohistochemistry, the specificity of the staining was ensured by replacing the primary antibody with 1% normal sheep serum. In all cases, no staining was observed.
For determination of apoptotic indices, bromodeoxyuridine-stained sections were also scored for apoptotic cells based on their morphology (pyknotic nuclei, cell shrinkage) [16]. Because this is a subjective method, samples were scored independently by two individuals, with similar results.
For staining of mast cells, samples were deparaffinized in xylenes, rehydrated through acetone and water, stained in 0.025% toluidine blue (Sigma) for 30s. Slides were washed in distilled water, dehydrated in acetone, cleared in xylenes, and mounted using Permount (Fisher). Mast cells were counted per high power (400×) field of view around the lesion.
Statistical analyses
For comparison of numbers of lesions, bromodeoxyuridine-labeling indices, and apoptotic indices at 20, 30, and 37 days after MNU treatment, t-tests using Bonferroni's correction were used. The data were also analyzed by square root transformation followed by t-tests using Bonferroni's correction. For comparison of cyclin D1 staining in Copenhagen and Wistar-Furth IDPs, a χ2 test was used, with the groups being IDPs that do not overexpress cyclin D1(- or +) and IDPs that do overexpress cyclin D1 (++). For comparison of percentages of cyclin D1-positive cells in Copenhagen and Wistar-Furth IDPs at day 37, a one-tailed t-test was used.