Lipidspot 610

A 2 mM chloroform/methanol stock solution of fluorescent AA {2-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino] AA (NBD AA), Avanti Polar Lipids} was kept at −80°C. Adult female and male flies (to allow for mating) younger than 2 weeks old were fed dry yeast for 48 h at room temperature or 24 h at 25°C in preparation for dissection. For in vivo supplementation, flies were fed supplemented yeast paste (1% sucrose, 5 µM NBD AA, dry yeast) overnight and follicles were dissected, fixed and stained as described in Staining Method 1. For in vitro supplementation, ovaries were dissected in maturation medium [Schneider's Drosophila medium, Sigma-Aldrich; 15% FBS, Gibco; 10 mg/ml insulin, Sigma-Aldrich; 1× penicillin/streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin, pH 6.95), Gibco], and then S10B follicles were isolated and incubated for 15 min in maturation medium supplemented with 5 µM NBD AA and 2.5 µl of LipidSpot 610 (1000×, Biotium). The medium was removed by aspiration and the follicles were washed once in fresh maturation medium for 5 min. The follicles were then transferred to a poly-D-lysine-coated, glass-bottom culture dish with 200 µl of maturation medium. Follicles were allowed to adhere to the culture dish for 5 min prior to imaging.

The following chemicals were used at the indicated concentrations: 20, 50, and 100 ng/ml Doxycycline (Sigma-Aldrich, D5207), 50 nM CHIR (Selleckchem, S2745), 3 µM PIM447 (Selleckchem, S7985), 4 µM GW6471 (Selleckchem, S2798), 100 µM Etomoxir (Selleckchem, S8244), 20 µg/ml cycloheximide (Selleckchem, S7418), 1:1000 LipidSpot488 (Biotium, 70065) or LipidSpot610 (Biotium, 70069), 167 nM SyTOX Green Nucleic Acid Stain (Fisher Scientific, S7020), 25 mM Glucose (Thermo Scientific, A24940-01), 10% Dialyzed FBS (Gibco, A33820-01).

Cells were plated in six-well plates containing microscope coverslips and treated as indicated. After treatment, cells were fixed with 4% formaldehyde for 20 min. and kept in a blocking solution (5% NGS and 0.3% Triton X-100 in PBS) for 60 min. Then, cells were incubated with anti-Catalase (CST, 12980), anti-PPARα (LifeSpan BioSciences, LS-B46), anti-Tom20 (CST, 42406) antibodies for 60 min. Following primary antibody incubation, cells were washed with 1X DPBS and incubated in secondary antibodies (Alexa Fluor 568 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit, 1:500 dilution) for 60 min. Finally, cells were mounted on glass slides with mounting media (CST, Prolong® Gold, 8961) containing DAPI. Images were taken at 60× magnification using a fluorescent microscope. For LD imaging, cells were seeded and fixed in 4% formaldehyde as described above followed by 30 min incubation in 1:1000 diluted LipidSpot 488 or LipidSpot610 (Biotium). After staining, cells were washed in 1X DPBS and mounted onto slides.

Serial sagittal sections (35 μm thick and separated by 280 μm) from E4FAD mouse brains were used for staining/immunostaining measures. For lipid staining, the sections were washed in TBS, mounted on glass microscope slides, and dried for 1 h. In the dark, 1X LipidSpot-610, “a fluorogenic neutral lipid stain that rapidly accumulates in lipid droplets where it becomes brightly fluorescent” (Biotium product sheet), was applied directly to the slides and incubated for 1 h (previously described [95 (link)]). The stained sections were imaged at 63X with a Zeiss Fluorescent Microscope, one field for subiculum (SB) and three fields for CX: visual CX, somatosensory CX, and frontal CX. Images were analyzed by counting the number of LD per nuclei (10–14 nuclei per field) and for the CX, averaged per mouse. For amyloid deposition, the sections were washed in TBS and stained with Thio-S. For Aβ deposition, astrogliosis, and microgliosis, sections were immunostained using MOAB-2, GFAP, and Iba1 antibodies, respectively (previously described [13 (link), 96 (link)]). Whole sections were imaged at 10X magnification with a Zeiss Fluorescence microscope and analyzed for area covered by Thio-S, Aβ, GFAP, and Iba-1 in CX using ImageJ software.

Seven days after adipogenic media and LIV treatment, cells were fixed and were stained with Oil Red O (Poly Scientific, Baywood, NY, USA, #k043), Lipid Spot 610 (Biotium, Fremont, CA, USA, #70069), and NucBlue Hoechst stain. Images were taken using a 20× objective and exported to quantify lipid droplet formation via a custom-made MATLAB program (MathWorks, Natick, MA, USA) previously published [63 (link)]. A minimum pixel intensity of 80 was used to isolate lipid droplet staining. The mean lipid droplet intensity per cell was calculated by dividing the sum of lipid droplet stain intensity by the nuclei count per image. For determining the effects of siLmna on lipid droplet formation, nuclear area, nuclear perimeter, and nuclear circularity, siCntl- and siLmna-treated cells were differentiated with previously stated adipogenic media and indomethacin (1 µg/mL) for 5 days. Cells were then stained for lamin A/C, as previously described, Lipid Spot 610, and NucBlue Hoeschst. Exported images were used to quantify lipid droplet formation, lamin A/C, nuclear area, nuclear perimeter, and nuclear circularity via the custom-made MATLAB program. A minimum pixel intensity of 80 was used to isolate lipid droplet staining and lamin A/C intensity.