Gestational Exposure to Ethane Dimethanesulfonate: MATERIALS AND METHODS(9)
The method for testis histology is a modification of the protocol by Klinefelter et al. and Parks et al.. The testes from males on PnD 1, 24, and 82 were removed and immersion fixed in 5% glutaraldehyde (#01909; Polysciences, Warrington, PA) in 0.05 M collidine buffer with 0.1 M sucrose overnight at 4°C. The tissue was rinsed twice with 0.2 M collidine buffer for 5 min. Testes were postfixed in 1% aqueous osmium tetroxide (#223A; Polysciences) in 0.05 M collidine buffer on ice for 1 to 2 h followed by a 5-min rinse in 0.2 M collidine. Dehydration consisted of two 5-min washes in 70%, 80%, and 95% ethanol on ice, and finally three 20 min washes in 100% ethanol at room temperature.
After two 10-min rinses in propylene oxide, a 1-h incubation in propylene oxide:Epon (1:1), and an overnight incubation (4°C) in 100% Epon, tissues were embedded in 100% fresh Epon at 60°C for 48 h. Sections (1 |xm thick) were cut with an RMS Ultramicrotome (MT-7; RMC Inc., Tucson, AZ) and stained with an aqueous solution of toluidine blue (#02205; Electron Microscopy Sciences, Fort Washington, PA). Images were captured with a Nikon Eclipse (E800, Nikon Instruments, Melville, NY) equipped with a digital SPOT camera (Southern Micro Instruments, Atlanta, GA) and corresponding SPOT RT version 3.2 software (Diagnostics Instruments, Sterling Heights, MI).