Oct4 c 10

Immunocytochemical analyses were performed on 10 μm frozen tissue sections. Sections were incubated with 10% normal goat serum (Thermo-Fisher) for 1 h, then overnight with primary antibodies to SUV39H2 (see above) or pan cytokeratin (F3418, Sigma Aldrich). Blastocysts fixed in 4% paraformaldehyde, washed with phosphate buffered saline (PBS, pH 7.4), and permeabilized in PBS containing 0.25% Triton X100. Following a blocking step in 10% normal goat serum for 30 min, blastocysts were incubated with the designated primary antibodies to caudal type homeobox 2 (CDX2, ab76541, Abcam, Cambridge, MA) or POU domain, class 5, transcription factor 1 (POU5F1, also called OCT4, C-10, Santa Cruz Biotechnologies, Santa Cruz, CA) overnight at 4°C. After washing with PBS, sections were incubated for 2 h with corresponding secondary antibodies: Alexa488-conjugated goat-anti-mouse immunoglobulin G (IgG, A32723, Thermo-Fisher) or Alexa 568-conjugated goat-anti-rabbit IgG (A11011, Thermo-Fisher). Nuclei were visualized with 4’,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Carlsbad, CA). Fluorescence images were captured using a Leica DMI 4000 microscope equipped with a charge-coupled device camera (Leica Microsystems GMbH, Welzlar, Germany).

Cells were fixed in 4% paraformaldehyde for 20 min at room
temperature and then incubated in blocking buffer (PBS containing
5%BSA and 0.2% Triton X-100) at 37 °C for 1 hr.
Cells were then incubated with primary antibodies diluted in blocking buffer
at 4 °C overnight followed by incubation with secondary antibodies
diluted in blocking buffer at 37 °C for 1 hr. Nuclei were stained
with Hoechst33342 (Invitrogen, 1:10000). Primary antibodies used include the
following: Oct4 (C-10, Santa Cruz, 1:200), Sox2 (Y-17, Santa Cruz, 1:200),
GATA4 (G-4, Santa Cruz, 1:200), Nanog (AF2729, R&D Systems, 1:200),
TUJ1 (Covance, Princeton, NJ, 1:500), Myosin (MF-20, DSHB, 1:50). Alexa
Flour fluorescent secondary antibodies (Invitrogen) were used at a 1:2000
dilution.

The following antibodies were used for flow cytometry, cell sorting and immunostainings respectively: mouse anti-human CD34-APC (Miltenyi and BD biosciences), CD34-PE (Miltenyi), mouse anti-human CD45-FITC (Miltenyi), CD45-APC (BD biosciences), CD45-VB (Miltenyi), mouse anti-human HLA ABC-VB (BD biosciences), CD31-FITC (BD biosciences), CD31-PE (BD biosciences), CD14-APC (Miltenyi), CD15-FITC (Miltenyi), mouse anti-human CD-133-APC (Miltenyi), mouse anti-human CD38-PE (BD biosciences), mouse anti-human CD90-FITC (BD biosciences), HLA-PE (BD biosciences), mouse APC isotype control (BD biosciences), mouse FITC isotype control (BD biosciences), mouse PE isotype control and mouse Vioblue isotype control (BD biosciences), OCT4 (C-10, SantaCruz, sc-5279, 1:100), LAMP-2 (1:50, Abcam), α-tubulin mouse IgG (1:500; Sigma), Anti mouse IgG-Cy3 (1:200; Jackson), Anti rabbit-IgG- Dylight 649 (1:200; Jackson), Alexa fluor 488-, 594- or 647-conjugated anti-rabbit or anti-mouse or anti-goat secondary antibodies (1/500, Jackson Immunoresearch), DAPI (5 mg ml⁻¹) (1:2000; Invitrogen).
For ChIP assay experiment, the following antibodies were used: anti-rabbit-IgGs (sc-2027 Santa Cruz Biotechnology), and rabbit anti-SOX2 (ab59776; Abcam). A total of 5 μg of each antibody was used for ChIP experiments.