Skip to main content
SpringerLink
Log in
Book cover

Immunocytochemistry and Related Techniques pp 281–297Cite as

Immunogold Electron Microscopic Quantification of Small Molecular Compounds and Proteins at Synapses and Other Neural Profiles

  • Protocol
  • First Online:

Part of the book series: Neuromethods ((NM,volume 101))

Abstract

This chapter describes procedures for quantification of postembedding labeling at brain synapses using computer-based tools . The postembedding electron microscopic immunogold method allows detection of epitopes with a resolution of about 20–30 nm. However, plasma membranes belonging to different cells and membranes of intracellular organelles can often be located even closer together. Localizing epitopes at such membranes can reliably be performed by using computer programs, such as ImageJ , which offers automated quantification of gold particles . The present chapter provides a practical description of how to use ImageJ and plug-ins to obtain an accurate representation of the subcellular localization of proteins and small molecular compounds .

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

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
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   109.99
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. Jourdain P, Bergersen LH, Bhaukaurally K et al (2007) Glutamate exocytosis from astrocytes controls synaptic strength. Nat Neurosci 10:331–339

    Article  CAS  PubMed  Google Scholar 

  2. Ottersen OP (1989) Postembedding immunogold labelling of fixed glutamate: an electron microscopic analysis of the relationship between gold particle density and antigen concentration. J Chem Neuroanat 2:57–66

    CAS  PubMed  Google Scholar 

  3. Bergersen LH, Morland C, Ormel L et al (2012) Immunogold detection of L-glutamate and D-serine in small synaptic-like microvesicles in adult hippocampal astrocytes. Cereb Cortex 22:1690–1697

    Article  CAS  PubMed  Google Scholar 

  4. Storm-Mathisen J, Ottersen OP (1990) Immunocytochemistry of glutamate at the synaptic level. J Histochem Cytochem 38:1733–1743

    Article  CAS  PubMed  Google Scholar 

  5. Gundersen V, Chaudhry FA, Bjaalie JG et al (1998) Synaptic vesicular localization and exocytosis of L-aspartate in excitatory nerve terminals: a quantitative immunogold analysis in rat hippocampus. J Neurosci 18:6059–6070

    CAS  PubMed  Google Scholar 

  6. Gundersen V, Fonnum F, Ottersen OP et al (2001) Redistribution of neuroactive amino acids in hippocampus and striatum during hypoglycemia: a quantitative immunogold study. J Cereb Blood Flow Metab 21:41–51

    Article  CAS  PubMed  Google Scholar 

  7. Morland C, Nordengen K, Larsson M et al (2013) Vesicular uptake and exocytosis of L-aspartate is independent of sialin. FASEB J 27:1264–1274

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Nordengen K, Heuser C, Rinholm JE et al (2013) Localisation of N-acetylaspartate in oligodendrocytes/myelin. Brain Struct Funct. doi:10.1007/s00429-013-0691-7

  9. Bergersen LH, Storm-Mathisen J, Gundersen V (2008) Immunogold quantification of amino acids and proteins in complex subcellular compartments. Nat Protoc 3:144–152

    Article  CAS  PubMed  Google Scholar 

  10. Sogn CJ, Puchades M, Gundersen V (2013) Rare contacts between synapses and microglial processes containing high levels of Iba1 and actin: a postembedding immunogold study in the healthy rat brain. Eur J Neurosci 38:2030–2040

    Article  PubMed  Google Scholar 

  11. Stensrud MJ, Puchades MA, Gundersen V (2014) GABA is localized in dopaminergic synaptic vesicles in the rodent striatum. Brain Struct Funct 219:1901–1912. doi:10.1007/s00429-013-0609-4

  12. Mayhew TM, Mühlfeld C, Vanhecke D et al (2009) A review of recent methods for efficiently quantifying immunogold and other nanoparticles using TEM sections through cells, tissues and organs. Ann Anat 191:153–170

    Article  CAS  PubMed  Google Scholar 

  13. Blackstad TW, Karagulle T, Ottersen OP (1990) MORFOREL, a computer program for two-dimensional analysis of micrographs of biological specimens, with emphasis on immunogold preparations. Comput Biol Med 20:15–34

    Article  CAS  PubMed  Google Scholar 

  14. Larsson M, Broman J (2005) Different basal levels of CaMKII phosphorylated at Thr286/287 at nociceptive and low-threshold primary afferent synapses. Eur J Neurosci 21:2445–2458

    Article  PubMed  Google Scholar 

  15. Ruud HK, Blackstad TW (1999) PALIREL, a computer program for analyzing particle-to-membrane relations, with emphasis on electron micrographs of immunocytochemical preparations and gold labeled molecules. Comput Biomed Res 32:93–122

    Article  CAS  PubMed  Google Scholar 

  16. Anwar S, Peters O, Millership S et al (2011) Functional alterations to the nigrostriatal system in mice lacking all three members of the synuclein family. J Neurosci 31:7264–7274

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Berg LK, Larsson M, Morland C et al (2013) Pre- and postsynaptic localization of NMDA receptor subunits at hippocampal mossy fibre synapses. Neuroscience 230:139–150

    Article  CAS  PubMed  Google Scholar 

  18. Doig NM, Moss J, Bolam JP (2010) Cortical and thalamic innervation of direct and indirect pathway medium-sized spiny neurons in mouse striatum. J Neurosci 30:14610–14618

    Article  CAS  PubMed  Google Scholar 

  19. Janezic S, Threlfell S, Dodson PD et al (2013) Deficits in dopaminergic transmission precede neuron loss and dysfunction in a new Parkinson model. Proc Natl Acad Sci U S A 110:E4016–E4025

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Larsson M, Broman J (2006) Pathway-specific bidirectional regulation of Ca2+/calmodulin-dependent protein kinase II at spinal nociceptive synapses after acute noxious stimulation. J Neurosci 26:4198–4205

    Article  CAS  PubMed  Google Scholar 

  21. Larsson M, Broman J (2008) Translocation of GluR1-containing AMPA receptors to a spinal nociceptive synapse during acute noxious stimulation. J Neurosci 28:7084–7090

    Article  CAS  PubMed  Google Scholar 

  22. Medin T, Owe SG, Rinholm JE et al (2011) Dopamine D5 receptors are localized at asymmetric synapses in the rat hippocampus. Neuroscience 192:164–171

    Article  CAS  PubMed  Google Scholar 

  23. Nemani VM, Lu W, Berge V et al (2010) Increased expression of α-synuclein reduces neurotransmitter release by inhibiting synaptic vesicle reclustering after endocytosis. Neuron 65:66–79

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Diggle PJ (2013) Statistical analysis of spatial and spatio-temporal point patterns. CRC, Boca Raton, FL

    Google Scholar 

  25. Illian J, Penttinen A, Stoyan H et al (2008) Statistical analysis and modelling of spatial point patterns, vol 70. John Wiley & Sons, Chichester

    Google Scholar 

  26. Larsson M, Sawada K, Morland C et al (2012) Functional and anatomical identification of a vesicular transporter mediating neuronal ATP release. Cereb Cortex 22:1203–1214

    Article  PubMed  Google Scholar 

  27. Stensrud MJ, Chaudhry FA, Leergaard TB et al (2013) Vesicular glutamate transporter-3 in the rodent brain: vesicular colocalization with vesicular γ-aminobutyric acid transporter. J Comp Neurol 521:3042–3056

    Article  CAS  PubMed  Google Scholar 

  28. Bergersen L, Waerhaug O, Helm J et al (2001) A novel postsynaptic density protein: the monocarboxylate transporter MCT2 is co-localized with δ-glutamate receptors in postsynaptic densities of parallel fiber-Purkinje cell synapses. Exp Brain Res 136:523–534

    Article  CAS  PubMed  Google Scholar 

  29. Larsson M, Morland C, Poblete-Naredo I et al (2011) The sodium-dependent inorganic phosphate transporter SLC34A1 (NaPi-IIa) is not localized in the mouse brain: a case of tissue-specific antigenic cross-reactivity. J Histochem Cytochem 59:807–812

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Ottersen OP (1987) Postembedding light- and electron microscopic immunocytochemistry of amino acids: description of a new model system allowing identical conditions for specificity testing and tissue processing. Exp Brain Res 69:167–174

    Article  CAS  PubMed  Google Scholar 

  31. Royet JP (1991) Stereology: a method for analyzing images. Prog Neurobiol 37:433–474

    Article  CAS  PubMed  Google Scholar 

  32. Ormel L, Stensrud MJ, Bergersen LH et al (2012) VGLUT1 is localized in astrocytic processes in several brain regions. Glia 60:229–238

    Article  PubMed  Google Scholar 

  33. Ormel L, Stensrud MJ, Chaudhry FA et al (2012) A distinct set of synaptic-like microvesicles in astroglial cells contain VGLUT3. Glia 60:1289–1300

    Article  PubMed  Google Scholar 

  34. Valtschanoff JG, Weinberg RJ (2001) Laminar organization of the NMDA receptor complex within the postsynaptic density. J Neurosci 21:1211–1217

    CAS  PubMed  Google Scholar 

  35. Bergersen L, Ruiz A, Bjaalie JG et al (2003) GABA and GABAA receptors at hippocampal mossy fibre synapses. Eur J Neurosci 18:931–941

    Article  PubMed  Google Scholar 

  36. Ji ZQ, Aas JE, Laake J et al (1991) An electron microscopic, immunogold analysis of glutamate and glutamine in terminals of rat spinocerebellar fibers. J Comp Neurol 307:296–310

    Article  CAS  PubMed  Google Scholar 

  37. Larsson M, Persson S, Ottersen OP et al (2001) Quantitative analysis of immunogold labeling indicates low levels and non-vesicular localization of L-aspartate in rat primary afferent terminals. J Comp Neurol 430:147–159

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden

    Max Larsson

  2. Department of Oral Biology, University of Oslo, Oslo, Norway

    Linda Hildegard Bergersen

  3. Department of Neuroscience and Pharmacology, Faculty of Health Sciences, Center for Healthy Aging, University of Copenhagen, Copenhagen, Denmark

    Linda Hildegard Bergersen

  4. Department of Anatomy, Institute for Basic Medical Sciences, University of Oslo, 1105 Blindern, Oslo, 0317, Norway

    Linda Hildegard Bergersen & Vidar Gundersen

  5. Department of Neurology, Oslo University Hospital, Oslo, Norway

    Vidar Gundersen

Authors
  1. Max Larsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Linda Hildegard Bergersen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vidar Gundersen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vidar Gundersen .

Editor information

Editors and Affiliations

  1. Department of Veterinary Sciences, University of Torino, Grugliasco, Torino, Torino, Italy

    Adalberto Merighi

  2. Department of Veterinary Sciences, University of Torino, Grugliasco, Torino, Torino, Italy

    Laura Lossi

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Larsson, M., Bergersen, L.H., Gundersen, V. (2015). Immunogold Electron Microscopic Quantification of Small Molecular Compounds and Proteins at Synapses and Other Neural Profiles. In: Merighi, A., Lossi, L. (eds) Immunocytochemistry and Related Techniques. Neuromethods, vol 101. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2313-7_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2313-7_15

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2312-0

  • Online ISBN: 978-1-4939-2313-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation