2.8. Confocal microscopy analysis

To further explore the process of GalNAc‐siRNA/DP7‐C complex transport through early endosomes, late endosomes, and lysosomes, the time‐dependent colocalization of different transport stages with specific markers, such as early endosome antigen 1 (EEA1) and lysosomal associated membrane protein 1 (LAMP1) for late endosomes, was evaluated. Hep3B cells were incubated with FAM‐GalNAc‐siRNA/DP7‐C or FAM‐GalNAc‐siRNA/Lipo 2000 for 4 hours or 22 hours, respectively. Then, the cells were fixed in 4% paraformaldehyde for at least 20 minutes at room temperature. After washing with PBS, the cells were permeabilized with 0.1% Triton for 15 minutes at room temperature. Then, the samples were blocked with 0.05% BSA/PBS blocking buffer for 20 minutes. The transfected cells were then incubated for 2 hours with anti‐EEA1 (1:100, Invitrogen) and anti‐LAMP‐1‐PE (1:100, Invitrogen) antibodies in PBS. Then, the cells were labeled with monkey anti‐rabbit PE‐conjugated secondary antibodies (1:100, for 1 hours, at room temperature). The coverslips were gently washed with distilled water and mounted onto slides using Antifade Mounting Medium with DAPI (Solarbio), and the samples were analyzed by laser confocal microscopy (Olympus).

LysoTracker Red (Beyotime Biotechnology) was used to detect lysosomes. After transfection 4 hours or 22 hours later, Hep3B cells were subjected to LysoTracker Red staining at 37°C for 2 hours. Then, the cells were fixed with 4% paraformaldehyde, sealed with Antifade Mounting Medium with DAPI (Solarbio) and imaged using laser confocal microscopy (Olympus).