Anti nanog mouse polyclonal antibody

Blastocysts were used for the immunofluorescence staining applied to evaluate the quality of the embryos [32 (link)]. Following three washes in PBS containing 0.1% polyvinyl alcohol (PBS-PVA), blastocysts were fixed for 60 min in PBS-PVA containing freshly prepared 2% (w/v) paraformaldehyde and 0.2% (v/v) Triton X at 24 °C. After fixation, oocytes were washed with PBS-PVA (three times for 15 min each) and oocytes were immersed in PBS-PVA containing 2.5% (v/v) Tween 20 (GE Healthcare Co., Chicago, IL, USA) for 2 min at 24 °C. Blastocysts were washed with PBS-PVA (three times for 15 min each) again, incubated for 2 h in PBS-PVA containing 1% (w/v) BSA (PBS-BSA) at 4 °C and thereafter in blocking buffer (PBS-BSA containing 10% (v/v) goat serum) for 40 min at 24 °C. The blastocysts were incubated in primary antibody overnight at 4 °C. The primary antibodies used were an anti-CDX2 rabbit monoclonal antibody (1:500; BioGenex, Fremont, CA, USA) to detect the TE cells and an anti-NANOG mouse polyclonal antibody (1:500; Abcam, Cambridge, UK) to detect the ICM cells. Alexa Fluor 568-labeled goat anti-rabbit IgG and Alexa Fluor 488-labeled goat anti-mouse IgG (both 1:500; Thermo Fisher Scientific, Waltham, MA, USA) were used as secondary antibodies to detect the ICM and TE cells, respectively. DNA was stained with DAPI (2 μg/mL; Molecular Probes, Eugene, OR, USA).