Skip to main content

Preparation of Herpes Simplex Virus-Infected Primary Neurons for Transmission Electron Microscopy

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1144))

Abstract

Transmission electron microscopy (TEM) provides the resolution necessary to identify both viruses and subcellular components of cells infected with many types of viruses, including herpes simplex virus. Recognized as a powerful tool in both diagnostic and research-based virology laboratories, TEM has made possible the identification of new viruses and has contributed to the elucidation of virus life cycle and virus–host cell interaction.

Whilst there are many sample preparation techniques for TEM, conventional processing using chemical fixation and resin embedding remains a useful technique, available in virtually all EM laboratories, for studying virus/cell ultrastructure. In this chapter, we describe the preparation of herpes simplex virus-infected primary neurons, grown on plastic cover slips, to allow sectioning of neurons and axons in their growth plane. This technique allows TEM examination of cell bodies, axons, growth cones, and varicosities, providing powerful insights into virus–cell interaction.

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Roingeard P (2008) Viral detection by electron microscopy: past, present and future. Biol Cell Eur Cell Biol Organ 100:491–501

    CAS  Google Scholar 

  2. Bozzola J, Russell LD (1999) Electron microscopy. Principles and techniques for biologist, 2nd edn. Jones and Bartlett Publishers, USA

    Google Scholar 

  3. Egerton RF (2008) Physical principles of electron microscopy. An introduction to TEM, SEM and AEM. Springer, New York

    Google Scholar 

  4. Griffiths G (1993) Fine structure immunocytochemistry. Springer, Berlin

    Book  Google Scholar 

  5. Saksena MM, Wakisaka H, Tijono B, Boadle RA, Rixon F, Takahashi H, Cunningham AL (2006) Herpes simplex virus type 1 accumulation, envelopment, and exit in growth cones and varicosities in mid-distal regions of axons. J Virol 80:3592–3606

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  6. Miranda-Saksena M, Boadle RA, Aggarwal A, Tijono B, Rixon FJ, Diefenbach RJ, Cunningham AL (2009) Herpes simplex virus utilizes the large secretory vesicle pathway for anterograde transport of tegument and envelope proteins and for viral exocytosis from growth cones of human fetal axons. J Virol 83:3187–3199

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  7. Aggarwal A, Miranda-Saksena M, Boadle RA, Kelly BJ, Diefenbach RJ, Alam W, Cunningham AL (2012) Ultrastructural visualization of individual tegument protein dissociation during entry of herpes simplex virus 1 into human and rat dorsal root ganglion neurons. J Virol 86:6123–6137

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  8. Tennyson VM (1970) The fine structure of the axon and growth cone of the dorsal root neuroblast of the rabbit embryo. J Cell Biol 44:62–79

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  9. Nakata T, Terada S, Hirokawa N (1998) Visualization of the dynamics of synaptic vesicle and plasma membrane proteins in living axons. J Cell Biol 140:659–674

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  10. Goldberg JL (2003) How does an axon grow? Genes Dev 17:941–958

    Article  PubMed  CAS  Google Scholar 

  11. deKort EJ, Gribnau AA, vanAanholt HT, Nieuwenhuys R (1985) On the development of the pyramidal tract in the rat. The morphology of the growth zone. Anat Embryol 172:195–204

    Article  CAS  Google Scholar 

  12. Zhou ZH, Dougherty M, Jakana J, He J, Rixon FJ, Chiu W (2000) Seeing the herpesvirus capsid at 8.5 A. Science 288:877–880

    Article  PubMed  CAS  Google Scholar 

  13. Holland DJ, Miranda-Saksena M, Boadle RA, Armati P, Cunningham AL (1999) Anterograde transport of herpes simplex virus proteins in axons of peripheral human fetal neurons: an immunoelectron microscopy study. J Virol 73:8503–8511

    PubMed Central  PubMed  CAS  Google Scholar 

  14. Grunewald K, Desai P, Winkler DC, Heymann JB, Belnap DM, Baumeister W, Steven AC (2003) Three-dimensional structure of herpes simplex virus from cryo-electron tomography. Science 302:1396–1398

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the Australian National Health and Medical Research Grants (402457 and 570849), the Westmead Millennium Institute, and the Westmead Medical Research Foundation.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Monica Miranda-Saksena .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Miranda-Saksena, M., Boadle, R., Cunningham, A.L. (2014). Preparation of Herpes Simplex Virus-Infected Primary Neurons for Transmission Electron Microscopy. In: Diefenbach, R., Fraefel, C. (eds) Herpes Simplex Virus. Methods in Molecular Biology, vol 1144. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0428-0_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-0428-0_15

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0427-3

  • Online ISBN: 978-1-4939-0428-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics