H 1000

After behavioral experiments, we performed transcardial perfusion with PBS followed by fixation with 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were post-fixed in 4% paraformaldehyde for an additional 12–18 h at 4° C. Before sectioning, brains were rinsed three times in PBS and embedded in 4% agarose-PBS. Then, 50-μm-thick slices were made using a vibrating microtome (Leica, VT100S). Sections were then suspended in blocking solution (10% Normal Goat Serum and 0.1% Triton-X100 in 1× PBS) for 1 h followed by overnight incubation at 4 °C with the primary antibody. Next, sections were washed with PBS, incubated for 1 h at room temperature with the secondary antibody at 1:500 dilution. For histological visualization of GCaMP6s, we used primary goat polyclonal anti-GFP antibody (1:500 dilution; Abcam, ab6673) and secondary donkey anti-goat Alexa Fluor 488 (1:500 dilution; Abcam, ab150129). Sections were then dry-mounted on slides using Vectashield (Vector Labs, H1000) before imaging. No immunostaining was performed for the visualization of FusionRed or tdTomato. Imaging was performed using an upright fluorescence macroscope (Olympus BX61). Images were acquired using Ocular Scientific Image Acquisition Software (Teledyne Imaging). Visualization and analysis were performed using ImageJ/FIJI software packages.

The formalin-fixed and paraffin-embedded liver tissues were cut into 4-μm sections and the expression of Bnip3 was determined in tissue of normal and fibrotic livers. Cells seeded on coverslips were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and counter-stained with antibodies against Bnip3, LC3B or vimentin. The coverslips were mounted onto slides in anti-fade mounting medium (H-1000, Vectorlabs, USA) and fluorescent images were captured using confocal microscopy (FV3000, Olympus, Japan).

Cultured neurons were fixed with 1% paraformaldehyde in PBS containing 4% sucrose for 5 min at room temperature. Without washing, neurons were then permeabilized and blocked simultaneously in 100% methanol for 7 min at − 20 °C. Primary antibodies against LC3A/B (#12741, Cell Signaling), p62 (ab56416, Abcam), lysosomal-associated membrane protein (LAMP1) (H4A3, Santa Cruz), and ubiquitin (P4D1, Santa Cruz) were added to blocking solution containing 0.1% gelatin, 0.3% Triton X-100, 16 mM sodium phosphate, and 450 mM NaCl and incubated overnight at 4 °C. After washing with PBS, coverslips were incubated with AlexaFluor488 (#4412, Cell Signaling) or AlexaFluor594 (#8890, Cell Signaling)-conjugated secondary antibodies for 1 h at room temperature and then washed extensively with PBS and distilled water. Subsequently, coverslips were mounted with mounting medium (H-1000, Vector Laboratories). The samples were imaged with a confocal laser-scanning microscope (Nikon, Japan) using a 60X oil lens and 488 nm and 594 nm emission lasers.

Cells were grown on glass coverslips and cultured/treated as described above. ARPE19 mito‐QC cells were fixed in 3.7% PFA at pH 7.0 in 0.2 M HEPES, counterstained with Hoechst 33342 and VECTASHIELD Antifade Mounting Medium H‐1000 was used to mount coverslips on slides. Images were acquired using an ANDOR Spinning Disc Microscope equipped with a Zyla camera (Plan Apochromat ×40 objective, NA 1.15). For high‐content temporal analysis of LD biogenesis, ARPE19 cells were seeded on Ibidi black wall m plates (24 well format) with culture and treatment conditions as described above. Samples were labelled with BODIPY and MitoTracker and fixed as described above, followed by imaging with a PerkinElmer Opera Phenix Platform. For analysis of LDs in ULK1 KO cells, images were acquired using a wide‐field Nikon Eclipse Ti wide‐field microscope using a Nikon Plan Apo ×60 oil immersion objective.

Oil Red O staining and hematoxylin staining were performed after immunocytochemistry, according to standard protocols. For Oil Red O staining, after the last step of perilipin immunocytochemistry, sections were washed in PBS and afterwards rinsed in 60% isopropanol. After incubating in Oil Red O (Sigma, O1391) working solution for 15 min, sections were rinsed in 60% isopropanol and water. Then, the sections were either mounted in mounting medium with DAPI (Vector Lab, H-1200) or stained in hematoxylin (Fisher Scientific, SH30-500D) for 2 min and then mounted in mounting medium (Vector Lab, H-1000). The percentage of Oil Red O⁺ area in each field was used to quantify the extent of adipogenesis. At least eight random fields per sample and four samples were used for quantification.