Autophagy pp 211-221 | Cite as

Improved Electron Microscopy Fixation Methods for Tracking Autophagy-Associated Membranes in Cultured Mammalian Cells

  • Ritsuko Arai
  • Satoshi WaguriEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1880)


Autophagy-related organelles, including omegasomes, isolation membranes (or phagophores), autophagosomes, and autolysosomes, are characterized by dynamic changes in lipid membranes including morphology as well as their associated proteins. Therefore, it is critical to define and track membranous elements for identification and detailed morphological analyses of these organelles. However, it is often difficult to clearly observe these organelles with good morphology in conventional electron microscopy (EM), thus hampering 3D analyses and correlative light-electron microscopy (CLEM). Here, we focus on describing fixation procedures using (1) ferrocyanide-reduced osmium for CLEM and (2) aldehyde/OsO4 mixture for detecting omegasome structures and isolation membrane-associated tubules (IMATs). These methods can be easily applied to cultured mammalian cells for conventional and cutting-edge EM analyses, leading to a better understanding of ultrastructural details in autophagosome formation.

Key words

Ferrocyanide-reduced osmium fixation CLEM Aldehyde/OsO4 mixture Autophagosome Omegasome Isolation membrane-associated tubule (IMAT) 



We thank all the members in our department for helpful discussions. This work was supported by JSPS KAKENHI Grant numbers 24390048 and 15H04670 (to S. Waguri).


  1. 1.
    Eskelinen EL, Reggiori F, Baba M, Kovacs AL, Seglen PO (2011) Seeing is believing: the impact of electron microscopy on autophagy research. Autophagy 7(9):935–956CrossRefGoogle Scholar
  2. 2.
    Karanasios E, Walker SA, Okkenhaug H, Manifava M, Hummel E, Zimmermann H, Ahmed Q, Domart MC, Collinson L, Ktistakis NT (2016) Autophagy initiation by ULK complex assembly on ER tubulovesicular regions marked by ATG9 vesicles. Nat Commun 7:12420. Scholar
  3. 3.
    Hayashi-Nishino M, Fujita N, Noda T, Yamaguchi A, Yoshimori T, Yamamoto A (2009) A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nat Cell Biol 11(12):1433–1437. Scholar
  4. 4.
    Yla-Anttila P, Vihinen H, Jokitalo E, Eskelinen EL (2009) 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy 5(8):1180–1185CrossRefGoogle Scholar
  5. 5.
    Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, Griffiths G, Ktistakis NT (2008) Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J Cell Biol 182(4):685–701. Scholar
  6. 6.
    Uemura T, Yamamoto M, Kametaka A, Sou YS, Yabashi A, Yamada A, Annoh H, Kametaka S, Komatsu M, Waguri S (2014) A cluster of thin tubular structures mediates transformation of the endoplasmic reticulum to autophagic isolation membrane. Mol Cell Biol 34(9):1695–1706. Scholar
  7. 7.
    Karnovsky MJ (1971) Use of ferrocyanide-reduced osmium in electron microscopy. In: Proc 14th Annual Meeting Amer Soc Cell BiolGoogle Scholar
  8. 8.
    Hoshino Y, Shannon WA, Seligman AM (1976) A study on ferrocyanide-reduced osmium tetroxide as a stain and cytochemical agent. Acta Histochem Cytochem 9:125–136CrossRefGoogle Scholar
  9. 9.
    Langford LA, Coggeshall RE (1980) The use of potassium ferricyanide in neural fixation. Anat Rec 197(3):297–303. Scholar
  10. 10.
    Schnepf E, Hausmann K, Herth W (1982) The osmium tetroxide-potassium ferrocyanide (OsFeCN) staining technique for electron microscopy: a critical evaluation using ciliates, algae, mosses, and higher plants. Histochemistry 76(2):261–271CrossRefGoogle Scholar
  11. 11.
    Yla-Anttila P, Vihinen H, Jokitalo E, Eskelinen EL (2009) Monitoring autophagy by electron microscopy in Mammalian cells. Methods Enzymol 452:143–164. Scholar
  12. 12.
    Biazik J, Yla-Anttila P, Vihinen H, Jokitalo E, Eskelinen EL (2015) Ultrastructural relationship of the phagophore with surrounding organelles. Autophagy 11(3):439–451. Scholar
  13. 13.
    Hua Y, Laserstein P, Helmstaedter M (2015) Large-volume en-bloc staining for electron microscopy-based connectomics. Nat Commun 6:7923. Scholar
  14. 14.
    White DL, Mazurkiewicz JE, Barrnett RJ (1979) A chemical mechanism for tissue staining by osmium tetroxide-ferrocyanide mixtures. J Histochem Cytochem 27(7):1084–1091. Scholar
  15. 15.
    Audrey MG (1975) Fixation, dehydration and embedding of biological specimens. In: Audrey MG (ed) Practical methods in electron microscopy, vol 3. North-Holland Publishing Company, AmsterdamGoogle Scholar
  16. 16.
    Hayat M (1981) Fixation for electron microscopy. Academic Press, New YorkCrossRefGoogle Scholar
  17. 17.
    Yabashi A, Uemura T, Waguri S (2014) Optimal temperature in applying a fixative mixture of aldehyde and osmium tetroxide to the observation of isolation membrane-associated tubules. J Electr Microsc Technol Med Biol 28(1):9–11Google Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Anatomy and HistologyFukushima Medical University, School of MedicineFukushimaJapan

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