In Situ Hybridization
In situ hybridization was performed as described previously. Frozen sections (10 ^m) were mounted onto poly-L-lysine-coated slides and stored at -70°C until used. After removal from -70°C, the slides with uterine sections were placed on a slide warmer (37°C) for 2 min and then fixed in 4% paraformaldehyde in PBS for 15 min at 4°C. After prehybridization, uterine sections were hybridized to 35S-labeled LIF sense or antisense cRNA probes for 4 h at 45°C. After hybridization and washing, the slides were incubated with RNase A (20 ^g/ml) at 37°C for 15 min. RNase A-resistant hybrids were detected by autoradiography using Kodak (Eastman Kodak, Rochester, NY) NTB-2 liquid emulsion. The slides were poststained with hematoxylin and eosin. buy birth control online
A polyclonal antibody to rhLIF was used to detect im-munoreactive LIF in skunk uterine sections. This antibody (0.33 mg/ml) was preadsorbed with lyophilized skunk liver powder and used at a working dilution of 1:20. Uterine tissues were fixed in 2-4% paraformaldehyde (1-2 h, 4°C) and embedded in paraffin. Sections (6 ^m) were mounted on poly-L-lysine-coated slides and dewaxed in xylene.