Materials and Methods

Embryonic Sections Balb/c female mice were mated with Balb/c males and checked every 8 h for a vaginal plug, the appearance of which represented day O. Female mice were killed at different stages of gestation, and uterine horns were exposed by laparotomy and excised into Hanks' buffered saline (Washington University Tissue Culture Support Center). The embryos were freed from the extraembryonic membranes and fixed in 4% paraformaldehyde by immersion for paraffin embedding.

Immunohistochemistry Immunohistochemical staining was performed on sagittal sections of whole mouse embryos obtained as above. Sections were stained with an immunoperoxidase-based method at room temperature (Vectastain ABC Kit; Vector Labs, Burlingame, CA). Embryos were obtained at 11, 13, 16, 17, and 18 days of gestation. A total of 85 embryonic sections were examined. Embryos were fixed in phosphate-buffered saline containing 4% paraformaldehyde, dehydrated through a series of ethanol solutions, and embedded in paraffin. Sections (5 JLm) were deparaffinized, hydrated, and incubated with the indicated concentration of primary antibodies in diluent containing 25 mM Tris, 150 mM NaCI (TBS) and 0.05% normal goat serum for 1 h, rinsed with TBS, and incubated with biotinylated secondary goat anti-rabbit and goat anti-mouse antibodies. Afterwards, the sections were rinsed with TBS, incubated with avidin-biotin-horseradish peroxidase complex for 1 h, and rinsed and developed with 0.5% 3, 3'-diaminobenzidine-0.01% hydrogen peroxide solution in 0.1 M Tris (pH 7.2) for 7 min. Sections were counterstained with a solution consisting of 1% methyl-green plus 1% Alcian blue (Sigma Chemical Co., 51. Louis, MO) in 0.1 M acetate buffer (pH 6.5).