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The gonads were embedded in OCT medium (Tissue-Tek, Sakura Finetek, Torrance, CA) and frozen at —70°C. Serial sections of 10-^m thickness were used. After air drying, sections were treated with 1% Triton X-100 in water, rinsed with PBS, and blocked using 1% bovine serum albumin. The sections were then incubated overnight in Sox9 polyclonal antibody diluted 1:500. After washing in PBS, the sections were treated with fluorescein-goat anti-rabbit IgG (Sigma, St. Louis, MO) diluted 1:200 in blocking solution. RNA Isolation and RT-PCR Gonads were dissected from B6 and B6-Ytir embryos of 18.5 dpc and from animals of 30 and 60 dpp. The tissues were immediately frozen in dry ice and stored at —70° C. Detection was carried out in the two fetal gonads or single gonads of postnatal animals. Unilateral fetal gonads from hermaphrodite mice were pooled as two ovaries or two testes. A DNA probe containing a 243 bp fragment of p-actin was used as the internal expression control. Total RNA was isolated from gonads using Trizol reagent (Life Technologies Invitrogen, Carlsbad, CA); based on the method of Chomczynski and Sacchi, a few modifications were introduced to improve the RNA isolation, the volume of chloroform was increased to double, and the time of precipitation in isopropanol was increased from 10 minutes to overnight.