Cells were grown in chamber slides, fixed, and permeabilized, and then utilized to perform immunofluorescent staining with a rabbit anti-Sox2 antibody (#3579) and rabbit anti-Nanog antibody (#4903) (Cell Signaling Technology) overnight at 4°C, followed by goat anti-rabbit Alexa Fluor®488 antibody (A-11070, Life Technologies). Fluorescent cells were visualized and digital images were captured using an Olympus microscope.
For immunohistochemical (IHC) analyses, paraffin-embedded tissues were incubated with the indicated antibodies. The proportion score represents the estimated fraction of stained cells (0 = 0%, 1 = 1%–24%, 2 = 25%–49%, 3 = 50%–74%, and 4 = 75%–100%), while the intensity score represents their average staining intensity (0=no staining, 1=weak staining, 2=moderate staining, and 3=strong staining). The final staining score was determined by multiplying the intensity score by the proportion score. As a result, scoring was between 0 and 12.
For immunohistochemical (IHC) analyses, paraffin-embedded tissues were incubated with the indicated antibodies. The proportion score represents the estimated fraction of stained cells (0 = 0%, 1 = 1%–24%, 2 = 25%–49%, 3 = 50%–74%, and 4 = 75%–100%), while the intensity score represents their average staining intensity (0=no staining, 1=weak staining, 2=moderate staining, and 3=strong staining). The final staining score was determined by multiplying the intensity score by the proportion score. As a result, scoring was between 0 and 12.