Anti nanog antibody

Cells cultivated on Matrigel-coated coverslips were washed three times with PBS, fixed with 4% paraformaldehyde (Sigma-Aldrich, 158127) for 30 min, and blocked with 1% bovine serum albumin (BSA) in 0.1% Triton X-100 in PBS for 1 h. Coverslips were then incubated with a rabbit polyclonal anti-Nanog antibody (Santa Cruz Biotechnology, sc-33759) and a mouse polyclonal anti-Oct-3/4 (Santa Cruz Biotechnology, sc-5279) at a dilution of 1:200 overnight at 4°C. Donkey antirabbit Alexa 594 (Thermo Fisher Scientific, A21207) and donkey antimouse Alexa 488 (Thermo Fisher Scientific, A21202) were used as secondary antibodies at a dilution of 1:500 at room temperature for 1 h. Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI). Snapshots were taken with constant exposure time for individual channels using a fluorescent LSM700 microscope (×63 oil immersion objective; Carl Zeiss). Signal density per area was calculated for individual nuclei from raw images using ImageJ, and mitotic, overlapping, or otherwise irregular nuclei were left out of the analysis.

We took the lung tissues treated with 5-Fu after 24 h for immunofluorescence double staining. The paraffin-embedded tissues were cut into 4 μm thick slides, dewaxed in xylene, and dehydrated in graded alcohols. The antigen was retrieved by heating for 90 seconds in 0.01 mol/L citrate buffer (PH 6.0), followed by blocking administrated using nonspecific normal serum. The specimens were incubated separately with primary antibodies, anti-Nanog antibody (species: mouse, 1 : 100) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-SP-C antibody (species: rabbit, 1 : 100) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and anti-CCSP antibody (species: mouse, 1 : 100) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4°C. The two secondary antibodies were fluorescein isothiocyanate- (FITC-) conjugated anti-rabbit IgG and tetramethylrhodamine- (TRITC-) conjugated anti-mouse IgG in light-tight condition, and the nuclei were stained by DAPI (Sigma-Aldrich). For negative control, 1% BSA in PBS without primary antibody was used. The specimens were examined using a BX51 inverted epifluorescence microscope (Olympus).

Whole-cell lysates were dissolved in lysis buffer [150 mmol/L NaCl, 5 mmol/L EDTA, 1% Triton X-100, and 10 mmol/L Tris (pH 7.4)] with protease inhibitor (Roche; Mannheim, Germany) and subjected to electrophoresis on 4% to 15% Tris-glycine gels using 50–80 µg of protein per lane. Protein levels were first determined by the DC protein Assay (Bio-Rad; Hercules, CA). A Bio-Rad Trans-Blot system was used to transfer the proteins to Immobilon-P FL membranes (Millipore; Bedford, MA). Blots were incubated overnight at 4°C in 5% dry nonfat milk in TBS (150 mmol/L NaCl, 300 mmol/L KCl, 10 mmol/L Tris (pH 7.4), 0.01% Tween 20). Blots were incubated for 16 h at 4°C with anti-POU5F1 antibody at a dilution of 1∶500 (Santa Cruz; Santa Cruz, CA), anti-NANOG antibody at a 1∶100 dilution (Santa Cruz; Santa Cruz, CA), or antibody to β-actin (Santa Cruz; Santa Cruz, CA). A fluorescently labeled secondary antibody (Li-Cor; Lincoln, NE) was dissolved in 5% milk in the TBS-T buffer and incubated with the blot for 2 h at room temperature. After three 10 min washes, blots probed with fluorescently labeled antibody were imaged using an Odyssey Infrared Imager (Li-Cor; Lincoln, NE).