For COX-1 studies, an average of 10 pictures per section were taken, and the number of positive isolated cells in the subepithelial stroma were counted. For COX-2 and GM-CSF studies, an average of 20 pictures per section were taken. Areas of interest, either the conceptus, LE, or subepithelial stroma (S), were separately cropped and then submitted to densitometry analysis.
The amount of bi-nucleate cell migration is very extensive between d21P and d30P, rendering it difficult to distinguish between LE, hybrid syncytium, and trophoblastic cells. When the exact nature of the cells was uncertain, they were excluded from both groups (conceptus and LE). Color-cube-based segmentation was used to select only shades of red (AEC) in the area of interest. Integrated optical density (IOD) of the red staining was measured after standard OD calibration.
Assignment of animals to treatments was made at random. Data (dependent variables: intensity of COX-2/GM-CSF staining in the LE/S/conceptus, or number of COX-1-positive cells in the S) were subjected to least-squares factorial ANOVA by SuperANOVA software (Abacus Concepts, Berkeley, CA) followed by regression analysis or paired f-tests when main effects were significant (P < 0.05). In experiment 1, the effects of day within status (cyclic or pregnant) were examined by regression analysis. Data from days 7, 16, and 18 were also examined statistically across status. No significant difference was found between animals within the same group of days (animal; animal X horn, ANOVA, P > 0.05). After analysis, IOD/area data (±SEM) were converted to percentages (d7P or d18P = 100%) for ease of reporting. In experiment 2, main sources of variations included IFN-т treatment (0 or 1 mg total), side of the uterine horn (ipsi vs. contralateral), and associated interaction (treatment X horn). No significant difference was found between animals within the same treatment groups (animal; animal X horn, ANOVA, P > 0.1). IOD/area data (±SEM) were also converted to percentages (untreated control = 100%) after analysis.