Bodipy

Palmitic acid (P0500; Sigma–Aldrich, St. Louis, MO) and oleic acid (O-1008; Sigma–Aldrich) were used to induce stimulation and construct an in vitro cell model of NAFLD, as previously reported (23 (link)). For the control group, fatty acid–free BSA (0.5%; BAH66-0100; Equitech Bio, Kerrville, TX) was used. To visualize lipid droplets, cells were fixed using 4% paraformaldehyde and stained with BODIPY (D3922-10 mg; 1 μM in PBS; Thermo Fisher Scientific) for a duration of 3 min at room temperature. The cell nuclei were stained utilizing 4',6-diamidino-2-phenylindole staining. BODIPY images were acquired using a laser scanning confocal microscope (TCS SP8; Leica, Wetzlar, Germany).

For the visualization of lipid droplets, the third leaf from the non-necrotic tip region of both the transgenic line L13 and its corresponding WT was carefully excised. Subsequently, these leaf samples underwent fixation using a FAA buffer (4% formalin, 5% glacial acetic acid, 50% ethanol, 41% water, V/V) under vacuum conditions for a duration of 1 h. Following fixation, the samples were stained with a solution containing 12 μg/mL of BODIPY 493/503 (Invitrogen, Eugene, OR, USA) and 0.1% Triton X-100, employing vacuum assistance for 20 min. The imaging process was carried out using a Leica TCS SP5 laser scanning confocal microscope, employing an excitation wavelength of 496 nm for BODIPY and capturing emissions within the range of 505–583 nm. Additionally, chlorophyll autofluorescence was captured within the wavelength range of 661–800 nm.