To evaluate the effect of NTS on blastocyst cell numbers, blastocysts were fixed after 96 h culture and the control (0 nM) or 100 nM NTS samples were
immunostained. Briefly, blastocysts were treated with acetic Tyrode solution (pH 2.5) to dissolve zona pellucida and fixed with 4% paraformaldehyde-phosphate
buffered saline (PFA-PBS) with 0.2% Triton-X for 30 min at RT. After washing with 1% BSA-PBS, the specimens were blocked with 10% fetal bovine serum for 1 h and
incubated with anti-OCT4 mouse monoclonal antibody (1:100 dilution; #sc-5279, SantaCruz, Dallas, TX, USA) at 4°C overnight. Then specimens were treated with
donkey anti-mouse Alexa-555 antibody (1:200) and Hoechst 33452 (0.5 µg/ml) counterstaining for 1 h at RT. The washed samples were mounted on glass slides with a
small amount of 1% BSA-PBS, covered with a glass coverslip and pressed to spread the cells. Cell numbers were determined using an Olympus BX63 fluorescence
microscope (Tokyo, Japan). For counting, positive cells were defined by Hoechst staining, and cells in the inner cell mass (ICM) were those positive for OCT4,
calculated as a percentage of the total cells.
immunostained. Briefly, blastocysts were treated with acetic Tyrode solution (pH 2.5) to dissolve zona pellucida and fixed with 4% paraformaldehyde-phosphate
buffered saline (PFA-PBS) with 0.2% Triton-X for 30 min at RT. After washing with 1% BSA-PBS, the specimens were blocked with 10% fetal bovine serum for 1 h and
incubated with anti-OCT4 mouse monoclonal antibody (1:100 dilution; #sc-5279, SantaCruz, Dallas, TX, USA) at 4°C overnight. Then specimens were treated with
donkey anti-mouse Alexa-555 antibody (1:200) and Hoechst 33452 (0.5 µg/ml) counterstaining for 1 h at RT. The washed samples were mounted on glass slides with a
small amount of 1% BSA-PBS, covered with a glass coverslip and pressed to spread the cells. Cell numbers were determined using an Olympus BX63 fluorescence
microscope (Tokyo, Japan). For counting, positive cells were defined by Hoechst staining, and cells in the inner cell mass (ICM) were those positive for OCT4,
calculated as a percentage of the total cells.