Embryos were staged, collected, and fixed as described above. TUNEL labeling was carried out using the ApopTag Red In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA). Fixed embryos were washed in 1× PBS + 0.1% Tween 20 (PBTw), washed in equilibration buffer for one hour at room temperature, incubated in TdT reaction mix (70% reaction buffer to 30% TdT enzyme) for three hours at 37 C°, and then incubated in stop buffer for four hours at 37 C°. Next, embryos were blocked in BTN solution (1xBSS, 0.3% Triton X-100, and 5% normal goat serum) for one hour at room temperature, washed in 1xBSS, incubated in anti-dioxigenin and primary antibody overnight at 4 °C, washed with 1xBSS, incubated in secondary antibody for two hours at room temperature, washed, mounted and visualized as described above.

Top I-mediated ligation assay was carried out using the ApopTag ISOL Dual Fluorescence Apoptosis Detection Kit (DNase Types I & II) (Millipore, Billerica, MA, USA). Fixed embryos were rehydrated, washed with PBTw, incubated in the ISOL labeling reaction mixture in a dark humid chamber for 16 hours at 18 °C, and then blocked in BTN solution for one hour at room temperature. Embryos were then incubated with primary antibody overnight at 4 °C, washed with 1xBSS, incubated with secondary antibody for 2 hours at RT, washed, mounted, and visualized as described above.

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