Alexa fluor 647 labeled goat anti mouse igg

NPC1 or normal cells were seeded on 35-mm glass-bottomed dishes at a density of 1 × 10⁴ cells/dish and incubated overnight. After the medium was exchanged with fresh DMEM (900 μL), the treatment solutions (100 μL) were applied to the dish. After the cells were incubated with FITC-labeled HP-β-CD and HE-SS-PRX for 24 h, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min at room temperature, and permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Then, the cells were treated with mouse anti-human early endosome antigen 1 (EEA1) antibody (clone: 14/EEA1) (BD Biosciences) (1:100 dilution), mouse anti-human CD63 antibody (clone: H5C6) (BioLegend, San Diego, CA, USA) (1:200 dilution), or mouse anti-human lysosomal-associated membrane protein 1 (LAMP1) antibody (clone: H4A3) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:50 dilution) for 1 h at room temperature. After three washes with PBS, the cells were stained with Alexa Fluor 647-labeled goat anti-mouse IgG (Abcam, Cambridge, MA, USA) (1:1000 dilution) for 30 min at room temperature. Then, the cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Dojindo Laboratories) (1 μg/mL) for 10 min at room temperature. The CLSM observations were performed on a FluoView FV10i and the colocalization percentage was analyzed using FluoView Viewer (Olympus).