Morphologic Evaluation and Actin Filament: MATERIALS AND METHODS(4)

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Comparisonof Morphology andActin Filaments of In Vivo and InVitro Embryos (Experiment 2)

In experiment 2, in vitro-produced embryos were collected at 36 h (2- to 5-cell stages) and 6 days (blastocyst) after IVF. In vivo embryos were collected as mentioned above. Embryos were fixed in 3.7% paraformaldehyde in PBS for 2 h at room temperature. After fixation, embryos were treated with 1% (v:v) Triton X-100 in PBS for 6 h at room temperature or overnight at 4°C and then washed twice in PBS and cultured in a blocking solution (PBS containing 2% BSA and 150 mM glycine) for 30 min at room temperature. After being washed for another hour in PBS, the embryos were stained with 10 IU/ml rhodamine-phal-loidin (Molecular Probe, Eugene, OR) for 1 h at 39°C in PBS-Tween 20 (0.1%, v:v).

After washing twice in PBS-Tween solution for 2 h at room temperature, the embryos were stained with 100 nM YO-Pro-1 iodide (Molecular Probe) for 5-10 min for examination of nuclear status. Finally, the embryos were mounted on slides and examined by using confocal microscopy. buy cheap antibiotics

Confocal microscopy was performed using a Bio-Rad MRC-600 confocal laser scanning imaging system (Richmond, CA) equipped with a krypton argon ion laser, mounted on an Optiphoto II Nikon microscope (Garden City, NY) equipped with 40X or 60X objectives.