A blue filter was used on the microscope to filter out the blue counterstain of the sections. Standardized levels remained calibrated for all sections analyzed. Representative stained ovarian sections were chosen and three measurements of pixel density were made of the granulosa and thecal layers of every antral follicle in that section. For instance, in a section containing seven antral follicles, a total of 21 measurements of the granulosa cell layer and 21 measurements of the thecal layer were made. Means and standard error of the means were determined for pixel density measurements of granulosa and thecal layers from three individual ovarian sections of sesame control and 64-mg kg-1 day-1 MXC-treated mice.
Blood was collected during estrus from CD-1 mice after dosing and was subjected to enzyme-linked immunosorbent assay (ELISA) for measurement of estradiol levels. ELISA kits and reagents were obtained from Diagnostic Systems Laboratories, Inc. (Webster, TX). The assay was run using the manufacturer’s instructions and published methods. All samples were run in duplicate. The minimum detection limit, as stated in the instructions of the kit, was 7 pg/ml. The average intraassay coefficient of variation was 4.2% and the average interassay coefficient of variation was 8.2%.