Skip to Main content Skip to Navigation
Journal articles

Correlative fluorescence and electron microscopy of biocytin-filled neurons with a preservation of the postsynaptic ultrastructure.

Abstract : Several techniques enable to inject intracellularly neurons with dyes and to use light and electron microscopy to correlate the physiological data with the morphological properties of the neuron. However, the ultrastructure of the neuron is usually obscured by the injected dye thus notably precluding the analysis of the postsynaptic specialisation and that of the other organelles. To overcome this problem, we have developed a technique based on fluorophore- and ultra small gold-conjugated streptavidins. We report, that this method facilitates the identification of intracellular organelles of the biocytin-filled neuron and of postsynaptic densities. This method is valid for the study of early postnatal neurons that are particularly refractory to this type of analysis. The procedure introduced here consists of the following steps: (1) injection of biocytin into the neuron by a patch-clamp pipette, (2) aldehyde fixation, (3) reaction with a fluorophore-conjugated streptavidin, (4) analysis with a fluorescence microscope, (5) formation of avidin-biotin complexes (ABC), (6) reaction with an ultra small gold-conjugated streptavidin, (7) silver enhancement of gold, (8) postfixation with osmium tetroxide and embedding in resin, (9) ultrathin sectioning and analysis with an electron microscope. Using this method, we show that in early postnatal hippocampal neurons, that have been injected with biocytine, it is possible to determine the morphology of the dendritic and axonal trees (including very thin details such as spines and filopodia) and to identify the localisation of the symmetric and asymmetric synapses on dendrites of the injected neuron.
Document type :
Journal articles
Complete list of metadatas

Cited literature [3 references]  Display  Hide  Download

https://www.hal.inserm.fr/inserm-00484848
Contributor : Roman Tyzio <>
Submitted on : Wednesday, May 19, 2010 - 12:30:19 PM
Last modification on : Tuesday, November 10, 2020 - 3:09:50 AM
Long-term archiving on: : Thursday, December 1, 2016 - 1:50:51 AM

File

 Restricted access
To satisfy the distribution rights of the publisher, the document is embargoed until : jamais

Please log in to resquest access to the document

Identifiers

  • HAL Id : inserm-00484848, version 1
  • PUBMED : 12084567

Collections

Citation

Youri Morozov, Ilgam Khalilov, Yehezkel Ben-Ari, Alfonso Represa. Correlative fluorescence and electron microscopy of biocytin-filled neurons with a preservation of the postsynaptic ultrastructure.. Journal of Neuroscience Methods, Elsevier, 2002, 117 (1), pp.81-5. ⟨inserm-00484848⟩

Share

Metrics

Record views

130