Nonspecific background staining was reduced by microwaving the sections for two 5-min intervals in 0.05 M Tris-HCl, pH 7.6, allowing them to cool, and rinsing in PBS. The tissues were incubated for 30 min in 0.3% hydrogen peroxide in methanol, rinsed in PBS, and incubated for 20 min with normal rabbit serum (Vectastain ABC Goat kit); this was followed by endogenous avidin/biotin blocking (Vector Avidin Biotin kit). One section on each slide was exposed overnight at 4°C to primary antiserum (1:20 dilution in PBS). A negative control section on each slide was incubated with anti-LIF serum preadsorbed with an excess of rhLIF (5 ^g/ml in PBS) or incubated with goat IgG (1:20 dilution). Sections were washed in PBS and incubated for 10 min with anti-goat IgG biotinylated secondary antibody (Vectastain ABC Goat kit). After washing in PBS, sections were incubated with streptavidin-peroxidase conjugate, then with substrate-chromogen reagent (Vectastain AEC Goat kit), and counterstained with Mayer’s hematoxylin.
RIAs for plasma progesterone, estrogen, and PRL that were validated for use in the spotted skunk were performed as described previously. The interassay and intraassay coefficients of variation (mean ± SD) for the progesterone assay were 4.9 ± 6.1% and 4.6 ± 1.6%, respectively. Ovine (o)PRL (AFP 10789B from NIDDK-NIH, Rockville, MD) was iodinated by the chloramine-T method, and rabbit anti-oPRL (AFP C35810691 from NIDDK-NIH) was used at final tube dilution of 135 000. The intraassay variation (mean ± SD) between duplicate PRL samples was 8.1 ± 5.2%. Due to the limited quantity of plasma, estrogen was quantified in a single 1.0-ml sample from each animal. The interassay variation (mean ± SD) for the estrogen assay was 16.9 ± 8.9%. flovent inhaler
All data were analyzed by SPSS (SPSS Inc., Chicago, IL) or Statistica (Stat Soft, Inc., Tulsa, OK) software. Differences in uterine weight, LIF expression, and plasma hormone concentrations were analyzed by one-way ANOVA with post hoc multiple comparisons using a Bonferroni correction. Effects of hormone treatment on uterine weight and LIF expression were analyzed by Kruskal-Wallis one-way ANOVA. Comparisons of hormone-treated groups to controls were examined by post hoc Mann-Whitney U tests applying a Bonferroni correction to reduce the overall experimental type I error rate to 0.05.