Eugenia Morselli, Guillermo Mariño, Martin v. Bennetzen, Tobias Eisenberg, Evgenia Megalou, Sabrina Schroeder, Sandra Cabrera, Paule Bénit, Pierre Rustin, Alfredo Criollo, Oliver Kepp, Lorenzo Galluzzi, Shensi Shen, Shoaib Ahmad Malik, Maria Chiara Maiuri, Yoshiyuki Horio, Carlos López-Otín, Jens S. Andersen, Nektarios Tavernarakis, Frank Madeo, Guido Kroemer (2011) CIL:13917, Mus musculus, hepatocyte. CIL. Dataset

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

本研究采用表达GFP-LC3融合蛋白的转基因C57BL/6小鼠作为对照组，用于注射白藜芦醇和/或精胺。为避免死后自噬诱导，小鼠死亡后立即用4%多聚甲醛（质量/体积比，在pH 7.4的磷酸盐缓冲盐溶液中）进行灌注。随后，组织被采集并使用相同溶液进行≥4小时的进一步固定，接着用15%（质量/体积比，在磷酸盐缓冲盐溶液中）的蔗糖处理4小时，以及用30%（质量/体积比，在磷酸盐缓冲盐溶液中）的蔗糖过夜处理。组织样本被嵌入Tissue-Tek OCT复合物（Sakura）中，并储存在-70°C的温度下。使用冷冻切片机（CM3050 S；莱卡）制备了5微米厚的组织切片，空气干燥1小时，用磷酸盐缓冲盐溶液洗涤5分钟，室温下干燥30分钟，并用Vectashield抗褪色介质进行封片。使用配备有阿波色40× 1.15 NA浸没物镜的共聚焦荧光显微镜（TCS SP2；莱卡）捕获共聚焦荧光图像。图像使用莱卡DFC 350 FX 1.8.0相机通过LAS AF软件（莱卡）采集，并使用Adobe Photoshop（CS2版本）软件进行处理。具体而言，图片处理涉及代表性区域的裁剪以及对比度和亮度的线性调整，并使用Photoshop（对所有图片使用相同的调整参数）进行；未使用显式γ校正。图像：图9C左下角面板（肝脏/Co），见Morselli等人在《细胞生物学杂志》第192卷第615-629页的报道。