Bsas0

For immunocytochemistry, cells were fixed with 4% paraformaldehyde for 20 min at 4 °C. After washing with PBS, the cells were treated with 0.3% Triton X-100 in PBS for 10 min and blocked with PBS containing 3% bovine serum albumin (Bovogen, BSAS0.1) for 1 h at 25 °C. The cells were then treated with primary antibodies at the following concentrations: OCT4 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA, SC-5279), NANOG (1:500, Abcam, Cambridge, UK, ab80892), EOMES (1:500, Abcam, ab23345), GATA4 (1:200, Abcam, ab84593), tubulin beta III isoform (TUJ1; 1:500, Millipore, Burlington, MA, USA, MAB1637), smooth muscle actin (SMA; 1:500, Abcam, ab7817), SOX17 (1:500, R&D Systems, AF1924), and CDX2 (1:1250, Abcam, ab76541). The following day, the primary antibodies were removed and the cells washed thrice with PBS for 10 min. Finally, the cells were labeled with fluorescence-conjugated secondary antibodies to detect the primary antibodies at the following concentrations: Alexa Fluor 488 (1:500) and Alexa Fluor 568 (1:500). Lastly, they were treated with DAPI or Hoechst in 0.3% Triton X-100 in PBS for 3 min at room temperature and washed.

For immunochemistry, the cells were cultured until 70–80% confluency was achieved. The cells were fixed in 4% paraformaldehyde at 4 °C for 30 min. Following fixation, the cells were washed with PBS and treated with 0.3% Triton X-100 in PBS for 10 min. Subsequently, a blocking step was performed in PBS containing 3% bovine serum albumin (Bovogen, BSAS0.1) at 1 h at 25 °C. The blocked cells were then treated overnight with the Ki67 (1:200; GeneTex, GTX16667) primary antibodies at 4 °C. After 16 h, the primary antibodies were eliminated by washing with PBS for 10 min, and the cells were labelled with fluorescent secondary antibodies (1:500, Abcam, Alexa Fluor 488). Finally, the cells were washed and treated with DAPI in 0.3% Triton X-100 in PBS for 4 min at 25 °C and washed (n = 4).

For immunofluorescence microscopy, the cells were cultured on coverslips submerged in a 60 mm² Petri dish. After the differentiation, the coverslip was collected, and the cells were fixed in 4% paraformaldehyde (#P6148, Sigma-Aldrich, St. Louis, MO, USA) for 30 min, permeabilized with 0.5% Triton X-100 (#TR1020-500, Biosesang, Sungnam, Korea) for 15 min, and then blocked with 5% BSA (# BSAS 0.1, Bovogen, Keilor East, Australia) prepared in PBS for 1 h then incubated with PTH antibody (1:200, LF-MA30443, AbFrontier, Seoul, Korea) for overnight at 4 °C. The coverslips were then incubated with secondary antibody (1:500, Alexa-488-conjugated goat anti-mouse IgG, # A-11001, Invitrogen, Waltham, MA, USA) for 1 h at room temperature. Visualization of images was carried out using a confocal microscope (LSM5 Pascall, Carl ZEISS, Oberkochen, Germany).