For immunocytochemical/stereological studies, testes were removed from 70% ethanol, rinsed two times in 95% ethanol for 15 min, three times in 100% ethanol for 20 min, and two times in 100% xylene for 30 min. Tissue was incubated in a 1:1 xylene:paraffin (Paraplast Plus; Oxford Labwares, St. Louis, MO) bath at 60°C, two times for 45 min, and then three times for 30 min in a 100% paraffin bath at 60°C. Paraffin blocks were allowed to cool overnight and then stored at 4°C until sections (4 to 6 |xm) were obtained. After sectioning, the slides were held at room temperature overnight and then stored at 4°C until immunocytochemical analysis.
P450 side chain cleavage (P450scc) enzyme was immunolocalized to positively identify LCs. Paraffin sections of the testis were deparaf-finized via a graded ascending series of ethanol and xylene and microwaved at low power for 60 sec in 500 ml 0.1 M sodium citrate buffer (#2121; Polysciences) pH 6 for antigen retrieval. Sections were washed two times in PBS for 3 min, incubated for 20 min at room temperature in 30% H2O2 in methanol, and again washed two times in PBS. Sections were blocked for 1 h at 34°C with sterile PBS containing 1.0% protease-free BSA (#A-3294, Sigma), 10% normal goat serum (#S-1000, lot #I 1013; Vector Laboratories, Inc., Burlingame, CA), and 0.3% Triton X-100 (Surfact-Amps X-100; #46472; Pierce, Rockford, IL).