Anti nanog af1997

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.

To demonstrate pluripotency markers in the undifferentiated cells, live cells were fixed and permeablized before incubated with primary antibodies, anti-Nanog (AF1997, R&D Systems), SOX2 (MAB2018, R&D), Oct4 (MAB4401, Millipore) and Tra-1-81 (560161, BD Biosciences) antibodies in 5% BSA overnight, followed by secondary antibodies conjugated with Alexa-Fluor-488 (A-11008, or A-11029, ThermoFisher Scientific). Slides were covered by anti-fade mounting medium with DAPI (H-1200, Vector Laboratories) and visualized under Zeiss Axioplan fluorescence microscope (Carl Zeiss, NY).
To detect the surface markers by flow cytometry, cells were prepared and stained as previously described8 (link)9 (link) before reading with the fluorescent-activated cell-sorting facility (FACSCalibur, BD) of phycoerythrin (PE)-conjugated SSEA4 (560128, BD Biosciences) and Tra-160 (560884, BD Biosciences), or allophycocyanin (APC) or PE-conjugated CD31 (555446, BD Biosciences), CD34 (555824, BD Biosciences), CD44 (559942, BD Biosciences), CD73 (550257 BD Biosciences), CD105 (17-1057, eBioscience) and CD146 (550315, BD Biosciences). Staining of intracellular protein osteocalcin (IC1419P, R&D) was performed according to the manufacturer’s recommended protocol. These flow cytometry data was analyzed with FlowJo software (Tree Star, Ashland, OR).

Normal rabbit IgG (12–370) and normal mouse IgG (12–371) were purchased from Millipore/Upstate. H3K79me3 (17–10,130), H3K36me3 (17–10,032), H3K27me1 (17–643), H3K4me3 (07–473), Acetyl-Histone H4 (17–630) and H3K9ac (17–658) antibodies were procured from Millipore. All of the above antibodies were used for ChIP analysis at 5 μg antibody per 30 μg chromatin. Oct-3/4 (sc-8628) and Sox2 (sc-17,319) antibodies were procured from Santa Cruz Biotechnology. Anti-Nanog (AF1997) was purchased from R&D Systems. Anti-H3 (ab1791) was procured from Abcam. All of the above antibodies were used for immunoblot analysis at 1:1000 dilution.