Anti oct4

N-terminal C9ORF135 peptides were used to immunize two rabbits over the course of one month. The immunized sera were collected and purified with an affinity column (Epitomics Inc., Burlingame, CA, USA). The purified antibody was used for immunofluorescence analysis alongside antibodies against TUBB3 and MAP2 (Chemicon, Temecula, CA, USA), E-cadherin (BD Biosciences, San Jose, CA, USA), and anti-FLAG (Sigma). The antibodies to Nestin, Sox17, and KDR were purchased from Abcam (Cambridge, UK), and anti-OCT4 was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Alexa Fluor-labeled secondary antibodies were obtained from Invitrogen.
The digested H1 hESCs were washed twice with PBS plus 0.5% FBS. Then, the cells were stained with primary antibody against C9ORF135 (1:200) and the corresponding secondary antibody (goat anti-rabbit IgG). Approximately 10,000 cells were acquired with a BD FACSCalibur machine (BD Biosciences). Data were analyzed with FACSCalibur tools.

Cells were fixed with 4% PFA for 30 minutes and incubated in 1% Triton-X-100 for 15 minutes to permeate the cell membrane. Nonspecific binding was blocked with 1% BSA at room temperature for 1 hour. Proteins were detected with specific primary antibodies at 4 °C overnight. Primary antibodies were as follows: anti-NESTIN (1:100, MAB353; Millipore), anti-TuJ 1 (1:200, T2200; Sigma-Aldrich), anti-OCT4 (1:200, sc-8628; Santa Cruz), anti-NANOG (1:200, ab80892; Abcam), anti-phospho (Ser465/467)-SMAD2 (1:200, #3108; CST), and PAX6 (1:200, AB_528427; DSHB). After three washes with PBS, cells were incubated with corresponding secondary antibodies (1:1000; Jackson ImmunoResearch) for 1 hour. DNA was counterstained with Hoechst33342 (Invitrogen) for 5 minutes at room temperature. Immunofluorescent images were obtained on an Axioplan Zeiss microscope (LSM 780; Carl Zeiss). Quantitative analysis of immunofluorescent staining was performed using ImageJ software when the immunofluorescent images were obtained at the same exposure parameters.
For FACS analysis, cells were digested into single cells, followed by two washes in DPBS. The cells were then filtered through a 35-μm cell strainer cap (Falcon™ Cell Strainers, 352235). Sox1-GFP cells were sorted and counted by flow cytometry. Analysis was performed on a FACS-Canto flow cytometer (Beckman Coulter MoFlo™ XDP).

Mouse embryos were rinsed in Acid Tyrode’s solution, (Sigma Aldrich) and fixed in 3.7% formaldehyde on ice for 30 minutes. Primary antibodies used were anti-Oct4 (1:250, Santa Cruz Biotechnology) and anti-Cdx2 (1:200, BioGenex) and secondaries were Alexa555 and CruzFlur647 (see Table D in S1 Text). DNA was stained with Hoechst (2μg/ml, Sigma Aldrich). Embryos were placed on a glass slide coated with a 1% agarose pad and compressed to a 3:1 aspect ratio. All confocal images were acquired using a 63x 1.4 NA objective, on an Axioobserver Z1 Zeiss 780 confocal microscope with Zen2009 software. Z-stack images were acquired at 0.3μm intervals.

Antibodies used in this research are as follows: anti-OCT4 (sc-9081), anti-KLF4 (sc-20691), anti-GLIS1 (sc-67584), anti-c-MYC (sc-42), TRA-1-60 (sc-21705), SSEA1 (sc-21702), SSEA4 (sc-21704), anti-mouse (sc-2005), anti-rabbit (sc-2004) and anti-goat (sc-2020) from Santa Cruz; anti-SOX2 (AF2018) and anti-NANOG (AF1997) from R&D Systems; anti-LIN28A (#46020) from Abcam; anti-TRA-1-81 (09–0011) from Stemgent; Alexa Fluor 488 anti-mouse (A11029), Alexa Fluor 488 anti-rabbit (A11034) and Alexa Fluor 488 anti-goat (A11055) for immunostaining of iPS clones from Life Technologies. IRDye800 anti-mouse (610-132-121) for Odyssey imaging of iPSC colonies from Rockland.

Epithelial cells, oocyte-like cells, and colonies maintained on hAECs were fixed with 4% paraformaldehyde for 15 to 20 minutes at room temperature and permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature. Cells were then blocked with blocking solution for 30 minutes and incubated with anti-OCT4 (rabbit anti-human 1:200; Santa Cruz), anti-NANOG (rabbit anti-human, 1:200; Chemicon, Rolling Meadows, IL, USA), anti-DAZL (goat anti human, 1:500; Santa Cruz), anti-STELLA (goat anti-human, 1:200; Santa Cruz), anti-ZPC (rabbit anti-human, 1:200; Santa Cruz), anti-SCP3 (rabbit anti-human, 1:800; Abcam), anti-GDF9 (rabbit anti-human, 1:200; Millipore), anti-SSEA4 (mouse anti-human, 1:100; Millipore), anti Tra-1-60 (mouse anti-human, 1:100; Millipore), and anti Tra-1-81(mouse anti-human, 1:100; Millipore) antibody for 1 hour at room temperature. Cells were then probed with fluorescein isothiocyanate-labeled IgG (1:200; Santa Cruz) or Rodamine (TRITC)-labeled IgG (1:100; Invitrogen) and incubated at room temperature for another 20 minutes. The slides were then covered with mounting medium (glycerol diluted 3:1 in PBS; Vector Laboratories, Burlingame, CA, USA). Fluorescence images were obtained with a Leica DMI3000 microscope.

The primary antibodies used were as follows (company, catalogue number): anti-ERCC6 (Abcam, ab96098), anti-NANOG (Abcam, ab21624), anti-SOX2 (Santa Cruz, sc-17320), anti-OCT4 (Santa Cruz, sc-5279), anti-SMA (Sigma, A5228), anti-TUJ1 (Sigma, T2200), anti-FOXA2 (Cell Signaling Technology, 8186S), anti-CD90-FITC (BD Bioscience, 555595), anti-CD73-PE (BD Bioscience, 550257), anti-CD105-APC (BD Bioscience, 17-1057-42), anti-IgG-FITC (BD Biosciences, 555748), anti-IgG-PE (BD Biosciences, 555749), anti-IgG-APC (BD Biosciences, 555751), anti-Lamin B (Santa Cruz, sc-6217), anti-LAP2 (BD Bioscience, 611000), anti-Ki67 (ZSGB-BIO, ZM0166), anti-P16 (BD Bioscience, 550834), anti-γ-H2AX (Millipore, 05-636), anti-Nestin (Millipore, MAB5326), anti-PAX6 (Covance, PRB-278P), anti-CPD (Cosmo Bio, TMD-2), anti-cleaved PARP (Cell Signaling Technology, 9541), anti-β-Actin (Santa Cruz, sc69879), anti-GAPDH (Santa Cruz, sc-25778), and anti-hCD31 (BD Bioscience, 555445).

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.