Plan apochromat 63 1.40 oil dic m27 lens

Cells were fixed with 3% electron microcopy–grade paraformaldehyde for 10 minutes and washed twice with PBS. LDs were stained with HCS LipidTOX Neutral Lipid Stain (Thermo Scientific, H34476) at 1:500 dilution in PBS for 2 hours. Cells were washed twice with PBS, and nuclei were counterstained for 10 minutes with Hoechst 33342 (Thermo Scientific, H3570) at a dilution of 1:500. Images were captured using the laser scanning confocal inverted microscope LSM710 (Carl Zeiss) with a Plan-Apochromat ×63/1.40 oil DIC M27 lens, and 14-optical Z-sections at 1 μm intervals were captured. Images are maximal projections of the Z-stacks. LD quantification was performed using the Find Maxima plugin in ImageJ (NIH).

Genetically matched mutant/control pairs of mouse kidney epithelial cells were grown to confluency, then treated with 100 μM of Palmitic Acid (Cambridge Isotope Laboratories, Inc. DLM-215-1) and 10 μM of BODIPY® 558/568 C₁₂ (Thermo Fisher Scientific Inc. Catalog number: D38354, 4-Difluoro-5-(2-Thienyl)-4-Bora-3a,4a-Diaza-s-Indacene-3-Dodecanoic Acid) overnight. Cells were washed with PBS then stained with 100 nM of MitoTracker® Green FM (Thermo Fisher Scientific Inc. Catalog number: M751) for 30 min. Images were randomly taken by ZEISS LSM 700 using the Plan-Apochromat 63 ×/1.40 Oil DIC M27 lens. Quantification of lipid droplet size and number was done in 10 randomly selected fields in two cell lines using ImageJ. An investigator blinded to genotype adjusted image intensity thresholds to detect only lipid droplets prior to the analysis. The experiment was performed four times.