Anti nanog

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).

The following antibodies were used: anti-ALB (Abcam, ab8940, 1:400); anti-β-actin (Santa Cruz Biotechnology, sc-130301, 1:5 000); anti-caldesmon (Sigma-Aldrich, C4562, 1:300); anti-CD73 (BD Biosciences, 550741, 1:50); anti-CD90 (BD Biosciences, 555595, 1:100); anti-CD105 (eBioscience, 1-1057, 1:100); anti-CENPA (Abcam, ab13939, 1:400); anti-fibrillarin (Abcam, ab4566, 1:100); anti-FLAG (Sigma-Aldrich, M2, 1:2 000 for western blotting, 1:400 for immunofluorescence); anti-GAL4 (Abcam, ab14477, 1:1 000 for western blotting, 1:400 for immunofluorescence); anti-γH2AX (Millipore, 05-636, 1:400); anti-IgG-APC (eBioscience, 555751, 1:100); anti-IgG-FITC (eBioscience, 555748, 1:100); anti-IgG-PE (eBioscience, 555749, 1:100); anti-MAP2 (Sigma-Aldrich, m4403, 1:500); anti-NANOG (Abcam, ab21624, 1:250); anti-nestin (Millipore, MAB5326, 1:500); anti-NeuN (Millipore, ABN78 1:400); anti-OCT4 (Santa Cruz Biotechnology, sc-5279, 1:100); anti-Nucleolin (Abcam, ab22758, 1:200); anti-PAX6 (Covance, PRB-278P, 1:500); anti-SMA (Sigma-Aldrich, A5228, 1:100); anti-SM22 (Abcam, ab14106, 1:200); anti-SOX2 (Santa Cruz Biotechnology, sc-17320, 1:100); anti-Tuj1 (Sigma-Aldrich, T2200, 1:500); Alexa Fluor 555-conjugated wheat germ agglutinin (Thermo Fisher, W32464, 1:500). Other reagents, including deoxycytidine, hydroxyurea, NAC, nocodazole, and thymidine, were purchased from Sigma-Aldrich.

Alkaline phosphatase staining was performed with BCIP/NBT Alkaline Phosphatase Colour Development Kit (Beyotime) according to manufacturer’s instructions. For immunostaining, embryos or cells were fixed with 4% paraformaldehyde for 30 min and then permeabilized with 1% Triton X-100 for 30 min followed by blocking with 2% BSA (Sigma). Cells were incubated in primary antibody overnight at 4 °C and secondary antibody at 37 °C for 1 h. Primary antibodies were used that were anti-OCT-3/4 (Santa Cruz), anti-NANOG (Abcam), anti-SOX2 (Cell signaling), anti-SSEA1 (Santa Cruz), anti-SSEA4 (Santa Cruz), anti-E-CADHERIN (BD Bioscience), anti-KLF4 (Stemgent), anti-CDX2 (Biogenex).

Aggregates were fixed with 4% paraformaldehyde. The fixed specimens were cryoprotected with a serial treatment of 15% and 30% sucrose and embedded in tissue freezing medium. Frozen tissue blocks were sectioned into 10 or 12 μm cyrosections. For immunostaining, a 3% Goat or Horse Serum and 0.1% Triton-X100 solution was used for primary antibody incubation. An Alexa Fluor 488, 568, or 647 conjugated anti-mouse IgG or anti-goat IgG and an Alexa Fluor 568 or 647 conjugated anti-rabbit IgG (Invitrogen) were used as secondary antibodies. A DAPI counterstain was used to visualize cellular nuclei (ProLong Gold antifade reagent with DAPI, Life Technologies). Microscopy was performed on a Nikon TE2000 Inverted Microscope or an Olympus FV1000-MPE Confocal/Multiphoton Microscope.
The following antibodies were used: anti-E-cadherin (rabbit, Abcam; mouse, BD Biosciences); anti-Nanog (rabbit, Abcam); anti-Pax8 (rabbit, Abcam); anti-Pax2 (rabbit, Invitrogen; mouse, Abnova); anti-Sox2 (mouse, BD Biosciences); anti-myosin7a (rabbit, Proteus); anti-acetylated-α-Tubulin (mouse, Abcam); anti-TuJ1 (mouse, Covance); anti-Calbindin2 (mouse, Millipore); anti-Brn3A (mouse, Millipore); anti-Brn3C (mouse, Santa Cruz Biotechnology); anti-CtBP2 (mouse, BD Biosciences); anti-GFP (mouse, Santa Cruz); anti-GFP (rabbit, Abcam); anti-GFP (mouse, Life Technologies); anti-Espin (rabbit, Gift of Dr. James Bartles).

Total protein was extracted by lysis buffer containing protease inhibitors (Promega). Equal amounts of protein separated by 10% sulphate‐polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride (PVDF) membrane. After incubation with anti‐CD44 1:200, anti‐Oct3/4 1:200, anti‐Sox2 1:200, anti‐Nanog and anti‐SPOP antibody (Abcam) overnight at 4℃, membranes were washed three times in TBST for 5 minutes and subsequently incubated secondary antibody with conjugated IRDye800 (1:5000, Rockland) at room temperature for 2 hours. The bands were scanned using an Odyssey infrared imaging system (LI‐COR, Lincoln) and quantified.