Immunofluorescent Characterization of Differentiation

Characterization of the generated cells during differentiation stages was performed using immunofluorescence staining, as previously described (Akbari et al., 2019b (link); Karagonlar et al., 2020 (link)). Briefly, the cells were fixed in %4 paraformaldehyde (PFA; cat no: # 158127, Merck) for 20 min at room temperature, washed three times with 1× PBS, then permeabilized using 0.5% TritonX (cat no: #28313, Thermo Fisher Scientific). After 2 hours, blocking staining was carried out using the following primary antibodies: OCT3/4 (cat no: # 75463S, Cell signaling), PAX6 (cat no: # 60433S, Cell signaling), NESTIN (cat no: # 33475S, Cell signaling), GFAP (cat no: # 12389T, Cell signaling) and β-TUBIII (cat no: # 4466S, Cell signaling). Slides were visualized using a confocal microscope (cat no: # LSM880, Zeiss).