Gestational Exposure to Ethane Dimethanesulfonate: MATERIALS AND METHODS(5)
For immunohistochemical localization of P450 side chain cleavage enzyme in the LCs, adult testes (PND 53) were removed and immediately immersed in Bouin fixative (50 X the volume of the tissue). A 26-gauge needle was used to puncture the tunica albuginea (two nicks on each side of the testis) to facilitate penetration of fixative into the testis. The testes were fixed overnight at 4°C. The following morning, the testes were placed into cassettes and rinsed three times in 70% ethanol for 30 min on a stir plate at 4°C. Following a fourth overnight rinse, the tissue remained in 70% ethanol until paraffin embedding.
Whole-Body T Extraction
Whole-body T extraction was performed as described by Parks et al.. Briefly, 1 ml double-distilled deionized water (ddH20) was added to each 12 X 75 mm culture tube or 2 ml ddH20 to each 13 X 100 mm tube, each tube containing a single fetus, and the contents were homogenized with a Polytron homogenizer (Brinkman Instruments, Westbury, NY). Samples were double extracted with diethyl ether, and the ether phase was evaporated in a fume hood and stored at room temperature for no more than 2 wk until RIA analysis for whole-body T was performed.
The cauda epididymis of prepubertal (PND 24) and adult (PND 82) mice was removed, trimmed free of fat, and weighed. Each sample was homogenized in a 1-ml handheld glass homogenizer for 30 sec with 0.25 ml homogenization buffer containing 10 mM Tris (#161-0719; BioRad, Hercules, CA), 1 mM EDTA (#E-6758; Sigma, St. Louis, MO), 0.5% 3-[3-cholamidopropyl-dimethyl-ammonio]-1 propane sulfonate (CHAPS; #75621-03-3; Calbiochem, La Jolla, CA), and 0.25% octyl-beta-D-gluco-pyranoside (OBG; #494460; Calbiochem) pH 7.2.