Tunel enzyme solution

Dewaxed sections were rehydrated, and antigen retrieval achieved by incubating the sections with 15 μg/ml Proteinase K in 10 mM Tris-HCL at pH 7.5 (03115887001, Roche, Basel, Switzerland) for 30 min in a humidified chamber. The sections were washed three times in PBS for 10 min each, incubated with freshly prepared TUNEL reaction mixture (TUNEL Enzyme solution diluted 1:12.25 in TUNEL Label solution, (11684817910, Roche, Basel, Switzerland)) for 60 min at 37°C in a humidified incubator. Subsequently sections were incubated with anti-fluorescein antibody conjugated to horseradish peroxidise, TUNEL-POD (1772465, Roche, Basel, Switzerland) diluted 1:2 in PBS, for 30 min at 37°C in a humidified incubator. The sections were washed three times in PBS for 10 min each, incubated with DAB chromogen-substrate buffer solution (K3468, DAKO, Baar, Switzerland) for 5–10 min until a brown signal was detected and immersed in PBS to stop the reaction. The sections were washed for 2 min under running distilled water and counterstaining as above

To analyze blastocyst apoptosis, blastocysts were fixed with 4% (v/v) paraformaldehyde for 1 h at room temperature, washed with DPBS-PVA, and permeabilized with 0.1% (v/v) Triton X-100 in 0.1% (w/v) sodium citrate for 1 h at room temperature. After rinsing with DPBS-PVA, the embryos were stained with 45 ml of TUNEL-Label solution (Roche, Mannheim, Germany) supplemented with 5 ml TUNEL-Enzyme solution (Roche) for 1 h at 39°C in a dark, humidified atmosphere. Subsequently, nuclei were stained with 5 μg/ml Hoechst-33342 for 10 min blastocyst nuclei were analyzed by the method previously described under epifluorescence microscope (Kidson et al., 2004 (link)).

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.