BrdU incorporation assay
To detect DNA replication, zygotes were labeled at 22 h after hCG
injection (reaching the PN3 stage when the DNA replication began
actively) with 1 mmol/l BrdU in KSOM for 2 h. Then the embryos were
fixed in 4% PFA for 20 min and permeabilized for 20 min in 0.5% Triton
X-100. Then BrdU incorporation into DNA was detected using the primary
rat anti-BrdU (Abcam, ab6326, 1:200) antibody with a corresponding
secondary antibody conjugated with Alexa FITC goat anti-rat antibody
(ZSGB-Bio, ZF-0315, 1:1000).