Slides were incubated at room temperature for 30 min in rabbit anti-Cytochrome P450scc (#RDI-P450sccabr, Research Diagnostics, Inc.; Flanders, NJ), diluted 1: 200 in PBS containing 1.0% BSA, and then washed two times in PBS. Following a 1-h incubation at room temperature with biotinylated antirabbit IgG (H+L) (#BA-10000; Vector Laboratories) diluted 1:100 in PBS, sections were washed twice in PBS, incubated for 1 h with avidin-biotin peroxidase complex (Vectastain ABC reagent; Vector Laboratories), and washed twice with PBS. Bound antibody was localized with Vector DAB peroxidase substrate kit (#SK-4100; Vector Laboratories). Sections were washed in dH2O and counterstained with hematoxylin. Slides were rinsed in running tap water and dehydrated through a graded descending ethanol series before coverslipping with Vectamount (H-5000; Vector Laboratories).
Testis sections representing 9 and 10 testes from control and EDS exposed PND 53 testes were used for stereology. For each section, five distinct areas were selected in a uniform fashion beginning with the center and moving to each of the four corners. Images were captured with a digital SPOT camera (Southern Micro Instruments, Atlanta, GA) connected to a Nikon Eclipse E800 microscope equipped with a X40 objective. Images were analyzed with SPOT RT version 3.2 software. This software was used to determine diameters and areas of a traced image. For each of the five captured images, the number of immunostained cells (LCs) was counted and interstitial and tubular areas were delineated. We justified using the volume equation for a sphere to define the LC volume because the ratio of the longest to shortest diameter did not differ significantly (P < 0.05).