Ferrocyanide

A total of 27 (WT) and 29 (AMPK-KO) T cells were analyzed by transmission electron microscopy. Naive T cells were maintained 4 days in IL-7-supplemented medium and then fixed overnight in 2.5% glutaraldehyde (Electron Microscopy Sciences, UK) at 4 °C and post-fixed with 1% osmium tetroxide (Electron Microscopy Sciences, UK) and 1.5% ferrocyanide (Electron Microscopy Sciences, UK) in 0.15 M cacodylate buffer. The cells were further stained with 1% uranyl acetate (Electron Microscopy Sciences, UK), serially dehydrated and embedded in epoxy resin (Agar 100 resin; Agar Scientific, UK). Ultrathin 70-nm sections were produced with a Leica EM UC6 ultramicrotome and mounted on copper–Formvar–carbon grids (Electron Microscopy Sciences). Observations were made on a Tecnai 10 electron microscope (FEI, Eindhoven, Netherlands), and images were captured with a Veleta camera and processed with SIS iTEM sofware (Olympus, Germany).

WT zebrafish embryos (32 hpf) were fixed overnight in 2.5% glutaraldehyde (Electron Microscopy Sciences), 4% PFA at 4 °C and post-fixed with 1% osmium tetroxide (Electron Microscopy Sciences) and 1.5% ferrocyanide (Electron Microscopy Sciences) in 0.15 M cacodylate buffer. The embryos were further stained with 1% uranyl acetate (Electron Microscopy Sciences), serially dehydrated and embedded in epoxy resin (Agar 100 resin; Agar Scientific). Resin blocks containing the processed embryos were trimmed to reach the ROI, which was evaluated by toluidine staining of thin sections (15 μm). Ultrathin 70 nm sections were then produced with a Leica EM UC6 ultramicrotome and mounted onto copper-Formvar-carbon grids (Electron Microscopy Sciences). Observations were made using the Tecnai 10 transmission electron microscope (FEI), and images were captured with a Veleta camera and processed using SIS iTEM v.5.1 software (Olympus).