Immunostaining of Reprogrammed Cells

Reprogrammed cells were gently washed with DPBS and fixed in 4% paraformaldehyde at room temperature for 20 min. The cells were washed again with 0.05% Tween-20 (Sigma-Aldrich) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) at room temperature for 15 min. After washing, they were blocked with 4% Donkey Serum at 4 °C overnight and wrapped in parafilm. Primary antibodies, SSEA4, OCT4, SOX2, TRA-1-60, and NANOG, were added to the cells and then, incubated at 4 °C overnight. The secondary antibodies, Alexa Fluor 594 anti-rabbit or Alexa Fluor 488 anti-rabbit antibodies (Life Technologies, Eugene, OR, USA), were supplemented after washing with 0.05% Tween-20. The samples were incubated at 4 °C overnight in the dark and stained with UltraCruz Aqueous Mounting Medium with DAPI (Santa Cruz Biotechnology, Dallas, TX, USA) for observation.
A CKX53 fluorescence microscope (Olympus, Tokyo, Japan) with a U-RFL-T fluorescence lamp (Olympus) was used to visualize the cells. Image analysis and colocalization studies were carried out using the Ocular Image Acquisition Software, version 2.0.1.496 (Digital Optics Limited, Auckland, New Zealand) [26 (link)].