The embryo was xed with 4% PFA for 15 min at room temperature and 0.3% Triton X-100 in PBS was added for 20 min to permeabilize the cell membrane. Then, the embryo was incubated with primary antibodies for TNF-α (1:100, Proteintech Inc., Rosemont, IL, USA) and CDX2 (1:100, Abcam, Inc., Cambridge, MA, USA) at 4 °C overnight. Subsequently, the embryo was incubated with Alexa Fluor 594conjugated goat anti-mouse IgG (H + L), (1:1000, Invitrogen, Inc., Carlsbad, CA, USA) and AlexaFluor 488conjugated goat anti-rabbit IgG (H + L) (1:1000, Invitrogen, Inc.) for 60 min at room temperature in the dark, followed by mounting with antifade mounting medium with DAPI (Beyotime, Inc., Shanghai, China).
The analysis was conducted using a confocal laser scanning microscope (Olympus FV1000, Olympus Corporation, Waltham, MA, USA) (Nikon N-STORM & A1, Nikon Corporation, Tokyo, Japan). The six embryos used for staining originated from the same volunteer.
The analysis was conducted using a confocal laser scanning microscope (Olympus FV1000, Olympus Corporation, Waltham, MA, USA) (Nikon N-STORM & A1, Nikon Corporation, Tokyo, Japan). The six embryos used for staining originated from the same volunteer.