2.7. Confocal analysis for intracellular localization

The KYSE520 cells were seeded at a density of 1 × 10⁴ cells·well⁻¹ and incubated for 24 h; they were then treated with 5 μg·mL⁻¹ LR004 and LR004‐VC‐MMAE for 30 min at 4 °C or for 0.5 and 10 h at 37 °C. After washing the wells with PBS, the cells were exposed on the chamber coverslip by the cytospin, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.2% Triton X‐100 for 5 min. LR004 and LR004‐VC‐MMAE were detected with an Alexa Fluor 488‐labeled goat anti‐human IgG (H+L) antibody. The lysosomes were labeled with a lysosomal‐associated membrane protein 1 (LAMP‐1) antibody followed by an Alexa Fluor 555‐labeled goat anti‐rabbit IgG (H+L) antibody. The cell nuclei were stained with 4′, 6‐diamidino‐2‐phenylindole (DAPI). Fluorescence images were acquired with the laser scanning confocal microscope (ZEISS LSM 710, Oberkochen, Germany).