Lsm5 exiter laser scanning confocal microscope

Cells were fixed for 30 min at room temperature in 4% paraformaldehyde. Then, the cells were washed three times with cold PBS for 3 min each at room temperature and incubated with blocking solution (5% BSA in PBS) for 1 h at room temperature. The cells were permeabilized with 0.1% Triton X-100 for 10 min and incubated overnight with primary antibodies at 4°C. The cells were rinsed with permeabilization buffer and then incubated with fluorescent-tagged secondary antibodies. The immunolabeled cells were mounted on slides and photographed using a Carl Zeiss LSM5 EXITER laser scanning confocal microscope.

In brief, cells were grown on cover glasses for 24 h then washed for twice with ice-cold PBS buffer. After fixing in 3% paraformaldehyde for 30 min at 4°C, the cells were washed with 50 mM NH4Cl and permeabilized with 0.1% Triton100 for 15 min at room temperature. The cells were then incubated with anti-RON, anti-β-catenin or anti-HIF-1α antibodies for 1 h at room temperature and labeled using Texas red-conjugated secondary antibodies. The immunolabeled cells were examined using Carl Zeiss LSM5 EXITER laser scanning confocal microscope. The immunofluorescence quantification for nuclear translocation was performed by manual counting (3 fields per section).

Huh7 cells were grown on coverslips and then fixed with 5% paraformaldehyde at 4 °C for 30 min. After washes with 50 mM NH4Cl, cells were permeabilized with 0.1% Triton X-100 for 15 min at room temperature. Nonspecific binding sites were blocked by 5% BSA for 1 h, and then were incubated with the primary ZCCHC13 antibody overnight. The Texas red-conjugated secondary antibody (ZsBio, Beijing, China) was added and incubated for 1 h at room temperature. After the incubation, mounting medium containing DAPI (ZsBio, Beijing, China) was applied to visualize the nuclei. Immunofluorescence staining was examined with a Carl Zeiss LSM5 EXITER laser scanning confocal microscope (Zeiss, Jena, Germany).