D4010

Three-day-old adult offspring were washed with M9 buffer three times before being stained with 30 µg/mL DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, US EVERBRIGHT, D4010) for ~3 h on a stir plate with continuous shaking at 350 rpm at room temperature. Animals were transferred to a new NGM plate after three washes with M9 buffer. Images were taken with an Axiocam 702 mono camera on a Zeiss LSM800 (Airyscan) confocal microscope using a 63× oil objective.

A confocal culture dish containing transfected cells was treated with 1,1'-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil, 3 μm, D4010, US Everbright, Inc.) to stain the cellular membrane. Following this, the cells were fixed using a 4% paraformaldehyde solution at room temperature for a duration of 30 min. The cells were then exposed to 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/ml, Invitrogen; Thermo Fisher Scientific, Inc.) for 5 min. Lastly, an appropriate volume of PBS buffer solution was introduced, and the cellular observations were made using a Zeiss LSM780 microscope.
Cells expressing eGFP and adhering to the dish were identified using a low power lens under light with an excitation wavelength of 488 nm. Subsequent observation of subcellular structures was carried out using an oil immersion microscope with a magnification of 630 times. The distribution of Dil was visualized by illuminating it with a laser excitation source at a wavelength of 549 nm. The distribution of DAPI was observed through excitation with a laser beam of 360 nm wavelength. Lastly, the distribution of the fusion protein eGFP-SCN3B was examined utilizing a laser source with an excitation wavelength of 488 nm.