4.4. Total Number of Cells, Cell Allocation, and TUNEL Staining

Differential staining was used to assess the total number of cells with DAPI staining and their allocation to the inner cell mass cells (ICM) by immunodetection of SOX2, and indirectly to trophectoderm (TE), by counting SOX2-non-expressing cells in expanded blastocysts generated by parthenogenesis (3 biological replicates, 8 total embryos per treatment), using a free plug-in available for ImageJ 1.53t software (National Institutes of Health, USA)

Immunostaining was performed as has been described previously [76], with a few modifications. Briefly, the embryos were collected and washed in PBS/PVA and fixed with 4% paraformaldehyde for 15 min and permeabilized with D-PBS with 0.1% sodium citrate containing 1% Triton X-100 for 30 min. After blocking for 2 h in D-PBS with 0.1% Triton X-100, 1% BSA, and 5% goat serum (Gibco, Auckland, New Zealand), embryos were placed in a primary antibody solution, consisting of blocking buffer, a mouse antibody anti-SOX2 (Abcam ab10005 at 1:500), or isotype control antibodies, overnight at 4 °C. After washing 3 times for 10 min and 3 times for 20 min each, embryos were incubated with secondary antibody (1:2000) Alexa Fluor 633-conjugated goat anti-mouse IgG (Life Technologies, Eugene, OR, USA, cat. # A-21052) at RT for 1 h. Embryos were then washed 3 times for 10 min and 3 times for 20 min each. For the TUNEL assay, embryos were incubated with labeling reagent according to the manufacturer’s instructions (In Situ Cell Death Detection Kit, Fluorescein, Roche Applied Science, Indianapolis, IN, USA). A positive control for TUNEL was carried out by treating embryos with 75.4 U DNase I for 15 min at 37 °C before the TUNEL assay, and a negative control was attained by incubating embryos with the fluorescent labeling reagent in the absence of the terminal transferase dUTP enzyme. Finally, embryos were mounted onto a glass slide with Prolong Gold Antifade with DAPI (Life Technologies, Eugene, OR, USA, cat. # {"type":"entrez-protein","attrs":{"text":"P36935","term_id":"549826","term_text":"P36935"}}P36935) and evaluated using confocal microscopy (Olympus FV 1000 laser-scanning confocal microscope). This experiment was performed in 3 different biological replicates corresponding to independent ovary collections on different days.