The immunofluorescence analysis was performed as described previously22 (link). Briefly, for immunocytochemistry, cells were fixed with 4% paraformaldehyde (PFA,
Biosesang, Gyeonggi-do, Korea) in PBS for 1 h at 4°C and were permeabilized with 0.1%
Triton X-100 (Sigma) in PBS for 5 min. Then, the cells were blocked with blocking solution
(DAKO North America Inc., Carpinteria, CA) for 1 h at RT and were incubated with primary
antibodies overnight at 4°C. Secondary antibodies were incubated for 1 h at RT. The
following antibodies and dilutions were used: LHX1 (1:100; Santa Cruz); PAX2 (1:100;
Thermo Scientific); GATA4 (1:100; Santa Cruz); and FSHR (1:100, Santa Cruz). The secondary
antibodies were as follows: Alexa-488, Alexa-594, Alexa-488 (1:200, Life
Technologies).
For immunohistochemistry, the primary antibodies used were as follows: GFP (1:100; Abcam,
Boston, MA) and GATA4 (1:100; Santa Cruz). The secondary antibodies used were as follows:
Alexa-488; and Alexa-594 (1:100, Life Technologies). Immunofluorescence images were taken
using a confocal microscope (Carl Zeiss LSM 880, Oberkochen, Germany).