The “stemness” of iPS cells was estimated by examining the expressions of Oct3/4, Nanog, and SSEA-4 using immunostaining. Briefly, iPS cells were cultured in 4-well chamber culture slides (Nalge Nunc International) for 5 days, and then fixed with 1% formaldehyde for 10 min. After blocking, the cells were incubated with primary antibodies against human Oct3/4, Nanog, and SSEA-4 (R&D Systems, Inc.) for 1 hr and then with the appropriate Alexa 680-conjugated secondary antibodies for 20 min. The nuclei were stained with Hoechst 33258. Staining for the expression of ALP was performed using an Alkaline Phosphatase staining kit (Cosmo Bio Co., Ltd).
The expression levels of Oct3/4 and Nanog were further examined by Western blotting, as described previously9 (link)22 (link). Briefly, total protein was purified from iPS cells, separated using SDS-PAGE gels, and then transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies against Oct3/4, Nanog, or β-actin, followed by the appropriate horseradish peroxidase-conjugated secondary antibodies, and then visualized using an enhanced chemiluminescence detection kit (Amersham Biosciences).
The expression levels of Oct3/4 and Nanog were further examined by Western blotting, as described previously9 (link)22 (link). Briefly, total protein was purified from iPS cells, separated using SDS-PAGE gels, and then transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies against Oct3/4, Nanog, or β-actin, followed by the appropriate horseradish peroxidase-conjugated secondary antibodies, and then visualized using an enhanced chemiluminescence detection kit (Amersham Biosciences).
