Blastocysts
were subsequently incubated overnight at 4 °C in
PBS containing 10 mg/mL bovine serum albumin (blocking
solution), and then incubated in fluorescein-conjugated
2=-deoxyuridine-5=-triphosphate and terminal deoxynucleotidyl
transferase (TUNEL reagent; Roche
Diagnostics, Corp., Tokyo, Japan) for 1 h at 38.5 °C.
Embryos were counterstained with 50 g/mL propidium
iodide after RNase treatment (50 g/mL RNase for 60
min at room temperature) to label all nuclei.