Lsm 700 confocal laser scanning microscope

TSCs were plated on 35-mm ibidi μ-dishes and differentiated to either EVT or SCT. Once differentiation was complete, the cells were incubated with W6/32-488 conjugated antibody (Biolegend, 311413; 1:150 diluted in DMEM:F12) for 30 min at 37°C. Cells were washed twice with PBS and culture media was replaced before imaging. Images were captured with a Zeiss LSM 700 Confocal Laser Scanning Microscope with ZEN Microscope Software.

The organoids were washed with cell recovery solution (Corning, USA) before being fixed for 30 min in 4% paraformaldehyde. After permeation and blocking, primary antibodies against human GATA3 (1,100, Abcam, UK), TFA2A (1,100, Abcam, UK), TFA2C (1,100, Abcam, UK), beta-hCG (1,500, Abcam, UK), E-cadherin (1,250, Abcam, UK), HLA-G (1,50, Abcam, UK), and PAPPA2 (1,100, Abcam, UK) were added and incubated at 4°C overnight. The following day, the organoids were sequentially incubated with fluorescence-labeled secondary rabbit or mouse antibody. Phalloidin and DAPI were added accordingly. Subsequently, the stained organoids were imaged using a ZEISS LSM 700 Confocal Laser Scanning Microscope and ZEN imaging software.