Isopentane

Isolated plantaris or tibialis anterior muscles were frozen in liquid-nitrogen-cooled isopentane (Wako Pure Chemicals Industries, Osaka, Japan) for 1 min and then placed on dry ice for 1 hr to vaporize the isopentane. Transverse cryosections (10 μm) were stained with hematoxylin and eosin (H and E).
For immunohistochemical analyses, transverse cryosections (6 μm) were fixed with 4% paraformaldehyde (PFA) for 10 min. For embryonic myosin heavy chain (eMyHC) staining, sections were fixed with cooled acetone for 10 min at −20°C. Detailed information on antibodies used in this study is provided in Key Resource Table. For mouse anti-Pax7 and -eMyHC staining, a M.O.M. kit (Vector Laboratories, Burlingame, VT, USA) was used to block endogenous mouse IgG before reacting with the primary antibodies. The signals were recorded photographically using a BZ-X700 fluorescence microscope (Keyence, Osaka, Japan).

A liver of the mouse was embedded in Neg-50 (Thermo Fisher Scientific) and allowed to stand at 4 °C for 18 h. Frozen blocks were prepared using isopentane (FUJIFILM Wako Pure Chemical, Osaka, Japan) immersed in liquid nitrogen. The frozen block was sliced into 7 μm thick tissue sections using a cryostat HM550OVP (Thermo Scientific Microm) and a MAS-coated slide glass (MATSUNAMI, Kishiwada, Osaka, Japan) was pressed against the tissue section for its attachment on the glass. The frozen tissue section on the glass was washed with water and immersed in Meyer-hematoxylin solution (FUJIFILM Wako Pure Chemical) for 10 min for nuclear staining. Subsequently, it was washed with running water for 5 min and immersed in an eosin solution for 2 min to stain the cytoplasm. It was rinsed with water for about 5 s to wash off excess eosin solution, and soaked in 70 (v/v) % ethanol for 5 min, then in each of 90, 95, and 100 (v/v) % ethanol for 2 min in this order. After that, it was soaked in xylene for 5 min twice, and encapsulated with a specimen-encapsulating agent New MX (MATSUNAMI), a xylene-based anti-fading agent.

Sepasol-RNA I Super G was purchased from Nacalai Tesque (Kyoto, Japan). Isopentane, formaldehyde, 2-mercaptoethanol, and 1% eosin Y solution were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). The PrimeScript RT Reagent Kit with gDNA Eraser was purchased from TaKaRa Bio (Shiga, Japan). SYBR FAST qPCR kits were purchased from Kapa Biosystems (Wilmington, MA, USA). Primary antibodies against AMPKα (#2532), phosphorylated Thr-172 AMPKα (#2535), Akt (#9272), phosphorylated Ser-473 Akt (#4058), forkhead box protein O1 (FOXO1) (#2880), phosphorylated Ser-256 FOXO1 (#9461), FOXO3a (#12829), and phosphorylated Ser-253 FOXO3a (#13129) were purchased from Cell Signaling Technology (Danvers, MA, USA); MEF2A (sc-313), PGC-1α (sc-517380), and Sp1 (sc-14027) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA); and α-tubulin (#017-25031) was obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). For secondary antibodies, goat anti-mouse IgG H&L (HRP) (ab6789) and goat anti-rabbit IgG H&L (HRP) (ab6721) were purchased from Abcam plc. (Cambridge, UK), and goat anti-rabbit IgG-HRP (sc-2004) was purchased from Santa Cruz Biotechnology. Polyvinylidene difluoride membrane (Immobilon-P, 0.45μm) for Western blotting was purchased from Merck (Darmstadt, Germany).