Transgenic C57BL/6 mice expressing GFP-LC3 fusion protein, uninjected as control for injection with Resveratrol and/or spermidine. To avoid postmortem autophagy induction, dead mice were immediately perfused with 4% paraformaldehyde (wt/vol in PBS, pH 7.4). Tissues were then harvested and further fixed with the same solution for ≥4 h followed by treatment with 15% sucrose (wt/vol in PBS) for 4 h and with 30% sucrose (wt/vol in PBS) overnight. Tissue samples were embedded in Tissue-Tek OCT compound (Sakura) and stored at −70C. 5-µm-thick tissue sections were generated with a cryostat (CM3050 S; Leica), air dried for 1 h, washed in PBS for 5 min, dried at RT for 30 min, and mounted with Vectashield antifading medium. Confocal fluorescent images were captured using a confocal fluorescence microscope (TCS SP2; Leica) fitted with an Apochromat 40× 1.15 NA immersion objective. Images were acquired with a camera (DFC 350 FX 1.8.0; Leica) using LAS AF software (Leica) and processed with Photoshop (CS2; Adobe) software. Specifically, picture processing involved cropping of representative areas and linear adjustments of contrast and brightness and was performed using Photoshop (with equal adjustment parameters for all pictures); no explicit γ correction was used. Image: Figure 9C, bottom left panel (liver/Co), in Morselli et al. J Cell Biol 192: 615-629