4 6 diamidino 2 phenylindole (dapi)

iPSC colonies were grown for 3 days on Matrigel™-coated coverslips suitable for immunofluorescence staining. The cells were fixed in 4% paraformaldehyde for 10 min at room temperature, rinsed twice with phosphate-buffered saline (PBS) and permeabilized in 0.5% Triton X-100 diluted in PBS for 10 min. Samples were then blocked with 2% FBS in PBS for 10 min at room temperature. The primary antibodies added were for typical pluripotency markers rabbit anti-Oct3/4 (1:100; Santa Cruz, CA, USA), goat anti-Nanog (1:20; R&D, Minneapolis, MN, USA), mouse anti-Sox2 (1:100; Millipore, Santa Cruz, CA, USA), mouse anti-TRA 1–60 (1:100; Millipore), mouse anti-TRA 1–81 (1:100; Millipore), mouse anti-SSEA4 (1:100; Hybridoma Bank, Iowa City, IA, USA) and kept at room temperature for 1 h. The secondary antibodies were as follows: donkey anti-rabbit Cye 3 (1:100; Chemicon) and donkey anti-mouse/goat Alexafluor 488 (1:100; Invitrogen, Carlsbad, CA, USA). Cells were also stained with DAPI (1:1000; Boehringer, Mannheim, Germany) for nuclei detection, diluted in blocking solution, and added in darkness at room temperature for 1 h. To stain the nucleus, 0.15% DAPI was added. The coverslips with the stained preparations were then mounted on glass. The staining was visualized using laser scanning confocal inverted microscope (Axio Observer.z1, Zeiss, Oberkochen, Germany).