In Situ Hybridization
In situ hybridization was performed with antisense 33P-labeled ribo-probes specific to HOXC10, HOXC11, HOXD10, and HOXD11. Probes are a gift of E. Boncinelli and have been previously characterized. Endometrium was fixed in 4% paraformaldehyde, cryoprotected in 30% sucrose, and then embedded in OCT compound (Miles Laboratories, Elkhart, IN). Ten-micrometer frozen sections were obtained and mounted on Vec-tabond-coated slides (Vector Laboratories, Inc., Burlingame, CA). Before use, sections were treated with 0.2 M HCl, Pronase (0.16 mg/ml), and 0.026 M acetic anhydride and were then dehydrated. Tissue sections were hybridized overnight with 3 X 106 cpm of each probe in 0.25 M NaCl, 0.01 M Tris-HCl (pH 7.5), 0.01 M NaPO4 (pH 6.8), 5 mM EDTA, Ficoll 400 (0.02%), polyvinylpyrolidone (0.02%), BSA Fraction V (0.02%), 50% formamide, 12.5% dextran sulfate, yeast tRNA (1.25 mg/ml), and 10 mM DTT.
Hybridization was performed in a humidified chamber for 16 h at 50°C. Slides were treated with RNase A at 37°C and were then washed for 16 h in 0.25 M NaCl, 0.01 M Tris-Cl (pH 7.5), 0.01 M sodium phosphate (pH 6.8), 5 mM EDTA, Ficoll 400 (0.02%), polyvinylpyrolidone (0.02%), BSA Fraction V (0.02%), and 50% formamide. Slides were dehydrated, dried, and dipped in K5D (Ilford Limited, Mobberley, Cheshire, United Kingdom) emulsion. Exposure was carried out at 4°C for 7-12 days, and slides were developed with D-19 (Eastman Kodak Co, Rochester, NY). Representative darkfield photomicrographs were taken at 20 X on an Olympus microscope (Olympus Corp., Lake Success, NY). Slides subsequently were counterstained with hematoxylin and eosin, and corresponding bright-field photomicrographs were taken.