Anti oct4

Cell lysates were separated by 10% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% fat-free milk in Tris-buffered saline, the following antibodies were used for western blotting: anti-Sox2 (1∶500), anti-ALDH1 (BD Biosciences, 1∶500), anti-Bmi1 (1∶500), anti-Oct4 (1∶500), anti-Nanog (1∶500), anti-vimentin (1∶500), anti-snail (1∶500), anti-β-catenin (1∶500), anti-E-cadherin (1∶500), and anti-β-actin (1∶1000) overnight at 4°C. All antibodies were obtained from Santa Cruz Biotechnology unless otherwise specified. After washing, the bound antibodies were visualized using horseradish peroxidase-conjugated anti-goat, ant-rabbit, or anti-mouse IgG (Thermo Fisher Scientific Inc., New York, NY) and the Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA) and subsequently visualized on X-ray films [21] (link).

For undifferentiated hESC cultures, cells were dissociated with Cell Dissociation Buffer (Life Technologies) for 5–10 minutes. Dissociated single cells were incubated with SSEA3 (rat anti-human IgM, Hybridoma Bank) or A2B5 (mouse anti-human IgM, Chemicon) antibodies for 40 minutes and then identified with fluorescent-conjugated secondary antibodies (Alexa 647-conjugated goat anti-rat IgM or Alexa 647-conjugated goat anti-mouse IgM, BD Biosciences). Anti-Oct4 (BD Biosciences) staining was identified using Alexa 647-conjugated goat anti-mouse IgG (BD Biosciences). Additionally, APC-conjugated c-kit (BD Biosciences) was also used for flow. Hematopoietic or neural cells derived from day 20 EBs were detected using CD45 (BD Biosciences), nestin (R&D Systems) antibodies, respectively. Following each staining, live cells were distinguished as 7-AAD negative (BD Biosciences). Flow analysis was performed on a FACS Calibur running Cell Quest Software (BD Biosciences). Postrun analysis was performed using FlowJo version 8.5.3 (Treestar) http://www.flowjo.com/ Ashland, OR, USA.

Adherent cells were cultured on ibidi μ-Slide and fixed in 4% PFA for 30 minutes at 4°C. Embryoid bodies were fixed in 4% PFA for 2h at 4°C and embedded in OCT compound for frozen sections. The samples were incubated with primary antibodies for overnight at 4°C and subsequently with fluorescence-conjugated secondary antibodies (Thermo Fisher Scientific) and DAPI for 1h at RT. The primary antibodies used were: anti-GFP (abcam ab13970, 1:1000), anti-PRDM1 (Cell Signaling Technology 9115, 1:200), anti-SOX17 (R&D AF1924, 1:500), anti-TFAP2C (Santa Cruz Biotechnology sc-8977, 1:200), and anti-OCT4 (BD Biosciences 611203, 1:500). Samples were imaged under Leica SP8 upright or inverted scanning confocal microscope and analysed using Volocity (6.3).

The subcutaneous tumors formed in mice were fixed in 10% phosphate-buffered formalin and embedded in paraffin. 4-µm thick sections were deparaffinized using xylene, and hydrated by a graded series of ethanol washes. Endogenous peroxidase activity was quenched by 10-min incubation in 3% H₂O₂. After incubation with blocking solution for 30 min, sections were incubated with primary antibody from BD Biosciences (anti-Snail, 1∶200 dilution; anti-E-cadherin, 1∶200 dilution, and anti-Oct4, 1∶300 dilution)for 1 h, a biotinylated secondary antibody for 20 min and then with streptavidin horseradish peroxidase (HRP) for 10 min. The antibody was visualized with diaminobenzidine (DAB) chromogen, and sections were counterstained with H&E.

Cells were seeded on 15-mm microscope cover glasses a day before the experiment. For immunostaining, cells were fixed with 4% paraformaldehyde, permeabilized (5% Triton-X, 20 mM HEPES, 3 mM MgCl₂·6H₂O, 300 mM sucrose), blocked (3% goat serum, New Zealand origin (16210072, Gibco, Thermo Fisher), 0.1% BSA in PBS), and incubated with primary antibodies overnight at 4 °C. Then, they were stained with Alexa Fluor-conjugated secondary antibodies goat anti-rabbit IgG (H+L) (Thermo Fisher) and goat anti-mouse IgG (H+L) (Thermo Fisher) at room temperature for 1 h. Nuclear staining was performed with DAPI. Antibodies for immunofluorescence are anti-Flag (rabbit) (GenScript A00170-40) and anti-Oct4 (mouse) (BD 611202).