Anti nanog

Explant and clonal cultures were fixed with 4% paraformaldehyde (PFA) for 1 h on ice. The cultures were immunostained with the primary antibodies for 16 h at 4°C, except in the case of immunostaining with anti-Nanog (Santa Cruz, Dallas, TX, USA, sc-30328), which was performed for 1 h at 37°C. The cultures were treated with secondary antibodies for 1 h at room temperature.
Neural tubes with attached DRG were dissected from E12 mouse embryos by the method described previously (Hall, 2006 ). The neural tubes with attached DRG were fixed with 4% PFA for 1 h on ice. The fixed tissues were immersed in gradually increasing concentrations of sucrose solution and embedded in OCT compound (Miles, Elkhart, IN, USA). Cryostat sections were cut at 10 µm and mounted on albumin-coated glass slides. The sections were immunostained with primary antibodies for 16 h at 4°C and then treated with secondary antibodies for 1 h at room temperature. Finally, the nuclei in cultures and sections were stained with 0.1 µg/ml DAPI (Dojindo, Kumamoto, Japan). DAPI nuclear staining was important for counting the exact number of immunoreactive cells in the DRG cell cultures and for judging cell death. Details of all antibodies used are listed in Table S2.

Immunofluorescence staining was carried out according to standard protocols. Primary antibodies used were anti-OCT4 (#sc5279 Santa-Cruz, Heidelberg, Germany, 1:200), anti-NANOG (#sc293121 Santa-Cruz, 1:200), anti-TRA1-60 (MAB4360 Millipore, Darmstadt, Germany, 1:250) and anti-SSEA-4 (MAB4304 Millipore; 1:250). Secondary antibodies used were goat anti-mouse IgG Alexa-546 (#A-11030 Invitrogen, Schwerte, Germany) and goat anti-mouse IgM Alexa-488 (#A-21042 Invitrogen). Nuclei were co-stained with 4,6-diamidino-2-phenylindole DAPI (1:2000). Alkaline Phosphatase staining was performed using the Alkaline Phosphatase Staining Kit II (#00-0055 Stemgent, Glasgow, United Kingdom) according to the manufacturer’s instructions.