Bx51 inverted epifluorescence microscope

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).

Immunofluorescence was performed as described previously [32 (link)]. Cells were fixed in 4% paraformaldehyde in 20 mM HEPES (pH 7.4) for 20 min, washed three times, and permeabilized with 1.0% Triton X-100 for 5 min. Cells were then incubated with rabbit polyclonal anti-Oct4 antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse polyclonal anti-β catenin antibody (1:200, Santa Cruz Biotechnology), mouse polyclonal anti-Fbxw7 antibody (1:200, Santa Cruz Biotechnology), mouse polyclonal antic-myc antibody (1:200, Santa Cruz Biotechnology), mouse polyclonal anti-Skp2 antibody (1:200, Santa Cruz Biotechnology) or mouse polyclonal anti-p27 antibody (1:200, Santa Cruz Biotechnology) overnight at 4°C before being washed three times and incubated with goat anti-rabbit conjugated secondary antibody or goat anti-mouse conjugated secondary antibody correspondingly for 1 h at room temperature in the dark. DAPI was used for nuclear counterstaining. The stained cells were mounted and viewed under a BX51 inverted epifluorescence microscope (Olympus, Tokyo, Japan).

Cells were fixed in 4% paraformaldehyde in 20 mM HEPES (pH 7.4) for 20 min, washed three times, and permeabilized with 1.0% Triton X-100 for 5 min. Cells were then incubated with rabbit polyclonal anti-Sox2 antibody, rabbit polyclonal anti-Oct4 antibody, rabbit RNA Polymerase II CTD Repeat Antibody (Phospho-Ser2) antibody, rabbit RNA Polymerase II CTD Repeat Antibody (Phospho-Ser5) antibody, rabbit polyclonal anti-β-catenin antibody, and mouse polyclonal anti-Nanog antibody for 1 h at room temperature before being washed three times and incubated with goat anti-rabbit conjugated secondary antibody and goat anti-mouse conjugated secondary antibody for 30 min at room temperature in the dark. DAPI was used for nuclear counterstaining. The stained cells were mounted and viewed under a BX51 inverted epifluorescence microscope (Olympus, Tokyo, Japan).