Coralite488 conjugated anti mouse igg

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).

Shikonin (purity above 98%) was purchased from Aladdin Biotechnology Co., Ltd. (Shanghai, China) and was dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO was controlled not to exceed 0.02% when diluted to the required concentration. Formic acid (FA) was purchased from Sigma-Aldrich (MO, United States). HPLC-grade methanol (MeOH) and acetonitrile (ACN) were obtained from VWR Chemicals (Paris, France). All the reagents and chemicals utilized in this study were of analytical grade. Human colon adenocarcinoma cell lines HT29, HCT116, SW480, and SW620 were purchased from KeyGen Biotech (Nanjing, China). The DAPI and Cell Counting Kit were purchased from Yeasen Biotechnology Co., Ltd (Shanghai, China). The β-tubulin antibody and CoraLite488-conjugated anti-mouse IgG were obtained from Proteintech (Rosemont, United States).