Nylon filters were cross-linked by UV light in a Strata-linker (Stratagene, La Jolla, CA) and prehybridized at 62°C for 2 h in 50% formamide, 5-strength saline sodium citrate (SSC; single-strength SSC IS 0.15 M sodium chloride, 0.015 M sodium citrate), 8-strength Denhardt’s solution (1.6 mg/ml Ficoll, 1.6 mg/ml polyvinylpyrrolidone, 1.6 mg/ ml BSA), 0.1% SDS, 10 mM EDTA, 25 mM Tris-HCl (pH 7.0), 250 mg/ml heat-denatured herring sperm DNA, and 250 mg/ml yeast tRNA. The hybridization was carried out in the same solution containing 2.5 X 106 cpm/ml of each probe for 16 h at 64°C. The filters were washed in doublestrength SSC, 0.1% SDS twice for 15 min at room temperature and then washed twice in 0.01-strength SSC, 0.1% SDS for 40 min at 66°C. Hybridizations using the p-actin probe was performed at 42°C as described. After hybridization, the filters were exposed to autoradiographic films or analyzed by Phosphorimager (Molecular Dynamics, Sunnyvale, CA). buy levaquin online
In Situ Hybridization of tPA and PAI-I mRNA in Cultured Granulosa Cells
To examine the expression and localization of tPA and PAI-I mRNA in cultured granulosa cells, 2 X 106 viable cells obtained from the eCG-primed ovaries were precultured for 6 h on poly-D-lysine-coated glass slides (22 X 22 mm) in a dish, in 1 ml of McCoy’s 5a medium containing 5% calf serum. After being washed, the cells were further incubated for 6 h in 1 ml fresh serum-free medium in the presence of LH (100 ng/ml) with or without PRL (1000 ng/ ml).