Apoptosis Analysis in Thawed Blastocysts

The TUNEL assay was performed as described by Lee et al. (21 (link)). To analyze apoptosis in the re-expanded and hatched blastocysts 24 h after freeze–thawing, the blastocysts were fixed with 4% paraformaldehyde for 1 h at 20–25°C and washed with DPBS. Permeabilization was performed using 0.1% Triton X-100 in 0.1% (w/v) sodium citrate for 1 h at 20–25°C. After washing with DPBS, the expanded blastocysts were stained with 45 mL of TUNEL-Label solution (Roche, Germany) containing 5 mL of TUNEL-Enzyme solution (Roche) for 1 h at 39°C in a dark, humidified atmosphere. Subsequently, the nuclei were stained with 5 μg/mL Hoechst-33342 for 10 min and the blastocysts were analyzed under an epifluorescence microscope.