Lipidtox deep red neutral lipid stain

Prior to flow cytometric sorting, cells were stained with LipidTOX™ Deep Red neutral lipid stain (Invitrogen, Cat. No. H34477, supplied as 1000X for standard assays) for 30 min at 37°C and DAPI (Axonlab, Cat. No. A4099.005, 5 mg/mL) for 10 min at room temperature. The cells were subsequently detached by incubating for 5 min in 0.05% trypsin (Gibco, Cat. No. 25300054) at 37°C. The OP9 cells were gently resuspended in PBS 1X before being filtered with a 100 μm cell strainer in FACS tubes.
After DLD sorting, the different sorted fractions were stained for LipidTOX™ Deep Red neutral lipid stain (Invitrogen, Cat. No. H34477, supplied as 1000X for standard assays) for 30 min at 37°C and DAPI (Axonlab, Cat. No. A4099.005, 5 mg/mL) for 10 min at room temperature prior subjected to Imagestream. We were limited to acquiring with ImageStream 100 viable single cells per sample due to time and volume handling limitations (Supplementary Fig. S4C).

Tissue preparation and processing for whole mount staining was performed as described previously^{42 (link)} with some adaptations. In short, dissected BAT was fixed in 4% paraformaldehyde and cut to small pieces (2 × 2 × 2 mm). After several PBS washing steps, autofluorescence was quenched by incubation with 5% glycine (45 min). For blocking and permeabilization, the tissue pieces were incubated with 0.3% Triton X100 + 0.1% sodium citrate in 3% BSA in PBS for 2 h. The tissues were rinsed twice with PBS. To stain apoptotic nuclei, the tissue pieces were incubated in TUNEL reaction mixture for 2 h at 37 °C in the dark (In Situ Cell Death Detection Kit, 11684795910, Roche). After PBS washing, nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) for 15 min (5 µg ml⁻¹). For staining of neutral lipids, tissue pieces were incubated with LipidTOX Deep Red Neutral Lipid stain (1:300 in PBS; H34477, Thermofisher) for 30 min. All steps were performed with continuous shaking. For microscopy, tissue pieces were transferred to a glass bottom dish (ibidi µ-Dish, ibidi GmbH) and imaged with a NikonA1 Ti confocal microscope (software NIS-Elements Advances Research, NIKON, RRID:SCR_014329).

Hearts were perfused with ice-cold PBS, dissected, embedded in O.C.T. compound (Tissue-Tek #4583), frozen in liquid nitrogen-cold Isopentane (Carl Roth #3927.1) and stored at -80°C. Sections were made with Cryostat (Leica #CM3050 S) at 7µm thickness and stored at -80°C. For immunofluorescence staining, fresh frozen sections were rinsed in PBS (Sigma-Aldrich #P3813-10PAK) for 5 minutes and incubated with Normal Goat Serum Block (Biolegend #927503) for 30 minutes at room temperature. Then, Sections were stained with primary antibodies recognizing F4/80 (Servicebio, GB113373, 1:5000), Spp1 (Servicebio, GB11500, 1:3000), Trem2 (Zenbio, #510482, 1:1000) overnight at 4°C in a humidified chamber. Secondary goat anti-rat Alexa Fluor 594 and LipidTox Deep Red Neutral Lipid Stain (Thermo Fisher) were applied for 1 hour at room temperature. Slides were mounted with ProLong Gold Antifade DAPI (Thermo Fisher).