Tcs spe ll

The immobilized (4% formaldehyde) and permeabilized (100% methanol) PC-9 cells (1 × 10⁶) were incubated with mouse monoclonal HE4 (sc-293473) and rabbit polyclonal EGFR (sc-120) in 5% BSA overnight at 4°C after blocking using 5% BSA. And then these cells were incubated with Alexa fluor 488 or 595 secondary antibodies (Invitrogen Molecular Probes, Carlsbad, CA, USA) after washing using PBS. The cells were photographed using a laser confocal microscope (TCS SPE ll, Leica, Germany) after washing and staining (DAPI).

MESO257 and MESO428 cells were seeded on laser confocal dishes (Solarbio, Beijing, China), then immobilized with 4% formaldehyde at RT for 15 min. The cells were washed three times with PBS (5 min/each time) and then permeabilized using 100% methanol at −20 °C for 10 min, and blocked using 5% bovine serum albumin (BSA) (Sigma, St Louis, MO, USA) and 0.5% Triton X-100 in PBS for 1 h at RT. Next, cells were incubated with primary antibodies for AXL (sc-166269, Santa Cruz Biotechnology. CA, USA) and p53 (#2527, Cell Signaling Technology, Shanghai, China) in 5% BSA overnight at 4 °C, and then rinsed in PBS (3 × 5 min, RT). The cells were then incubated with Alexa fluor 488 or 595 secondary antibody (Invitrogen Molecular Probes, Carlsbad, CA, USA) in 5% BSA in the dark for 1 h, at RT, and then rinsed in PBS (3 × 5 min, RT). Next, the cells were stained with DAPI (Beyotime, Shanghai, China) in the dark for 5 min at RT. Finally, immunofluorescence of the cells was visualized using a laser confocal microscope (TCS SPE ll, Leica, Germany). Experiments were performed in triplicate.