Lipi red probe ld03 10

To visualize the accumulation of lipid droplets, cells were stained with 1 μM Lipi-Red probe (LD03-10, Dojindo, Kumamoto, Japan) for 30 min. To confirm the colocalization of lipids and lysosomes, the cells were stained with 100 nM Lysotracker Blue DND-22 (L7525, Thermo Fisher Scientific, Waltham, MA, USA) or 100 nM Lysotracker DND-99 (L7528, Thermo Fisher Scientific, Waltham, MA, USA) for 15 min and 1 μM Lipi-Red probe (LD03-10, Dojindo, Kumamoto, Japan) for 30 min. To confirm the location of lysosomes, the hippocampal neurons were transfected with 2 μg of lamp1-mGFP (#34831, Addgene, Watertown, MA, USA) with 2 μL of lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA). The cells were then stained with anti-GFP antibody (A11122, Invitrogen, Carlsbad, CA, USA). The confocal microscopic images were acquired using an A1 Rsi/Ti-E confocal microscope (Nikon, Tokyo, Japan) with a 60 × oil-immersion lens. Z-stack image sequences were acquired at 0.4 μm intervals and converted with maximal intensity projection using NIS-element AR software (Nikon, Tokyo, Japan).