G7526

Aged animals were perfused as described above. Samples of approximately 1 mm³ were dissected from Harderian glands and subsequently fixed with 2.5% glutaraldehyde (Sigma, G7526) in 0.1 mol/l phosphate buffer, post-fixed with 2% osmium tetroxide (Sigma, 75633) in the same buffer, dehydrated in a graded series of ethanol, and embedded in Agar 100 epoxy resin (Agar Scientific, AGR1045). Ultra-thin sections were cut at a nominal thickness of 70 nm, post-stained with 2% uranyl acetate and lead citrate and inspected in a transmission electron microscope (Morgagni, FEI 268D TEM, UK) operated at 80 kV. Images were acquired using an 11-megapixel CCD camera from Olympus (Germany).

Lipid accumulation of adipocytes was visualized and quantified using an Oil Red O (ORO) Assay, a method that has been well characterized for staining and quantifying lipids in fat cells [98 ]. 3T3-L1 cells were washed with PBS and then fixed with a mixture of 1% Glutaraldehyde (Sigma-Aldrich®, G7526-10 mL) and 0.5% Paraformaldehyde (Sigma-Aldrich®, P6148-500 g). Once fixed, the cells were incubated in 60% isopropanol (Sigma-Aldrich®, 67–63-0), stained with the ORO solution (Sigma-Aldrich®, O1391-500 mL). The dye was extracted using 100% Butanol (Sigma-Aldrich®, B7906-500 mL) and quantified using a spectrophotometer (Multiskan Spectrum, Thermo Electron Corporation) at 510 nm.

The cell culture supernatant was centrifuged at 300g for 10 min at 4°C, followed by 2000g for 10 min at 4°C, and then 10,000g for 30 min at 4°C. The supernatant was then centrifuged at 100,000g for 70 min at 4°C, washed in PBS, and then centrifuged at 100,000g for 70 min at 4°C. Exosomes were resuspended in PBS. Sample aliquots of 6 μl were pipetted onto a 200-mesh copper grid (catalog no. 1GC200, Ted Pella Inc.) with carbon-coated formvar film and incubated for 1 hour. Excess liquid was removed by blotting. The exosome sample was fixed in 2% glutaraldehyde (v/v; G7526, Sigma-Aldrich, Saint Louis, MO) for 1 min. The grid was washed twice by brief contact with 100 μl of MilliQ water, each time for 1 min. Next, the grid was placed on 30 μl of 1.5% uranyl acetate (w/v) for 12 s, followed by using filter paper to remove excess liquid and air dry. JEOL 100CX TEM was used to acquire the images of the morphology of the exosome. The method was used for experiments in fig. S4B.