A total of 1 × 104 cells were plated in a glass bottom/confocal petri dish (Biosharp, Hefei, China) and cultured overnight. Cells were fixed by 4% paraformaldehyde solution (Beyotime) for 10 min and blocked with immuno-staining blocking buffer (Beyotime) for 60 min at room temperature, then incubated with mouse anti-EGFR (ProteinTech, 66455-1-lg) and rabbit anti-NK1R (Abcam, ab183713) overnight at 4 °C. The cells were then stained with coralite594-conjugated anti-rabbit IgG (ProteinTech, SA0013-4) and coralite488-conjugated anti-mouse IgG (ProteinTech, SA0013-1) for 2 h at room temperature. Nuclei were stained by DAPI (1 μg/ml, Sangon). Fluorescent images were captured by a microscope equipped with a laser-scanning confocal imaging system (Zeiss LSM800, German).
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