A rapid method for combined laser scanning confocal microscopic and electron microscopic visualization of biocytin or Neurobiotin-labeled neurons

Xue J. Sun, Leslie P Tolbert, John G Hildebrand, Ian A. Meinertzhagen

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Intracellular recording and dye filling are widely used to correlate the morphology of a neuron with its physiology. With laser scanning confocal microscopy, the complex shapes of labeled neurons in three dimensions can be reconstructed rapidly, but this requires fluorescent dyes. These dyes are neither permanent nor electron dense and therefore do not allow investigation by electron microscopy. Here we report a technique that quickly and easily converts a fluorescent label into a more stable and electron-dense stain. With this technique, a neuron is filled with Neurobiotin or biocytin, reacted with fluorophore-conjugated avidin, and imaged by confocal microscopy. To permit long-term storage or EM study, the fluorescent label is then converted to a stable electron-dense material by a single-step conversion using a commercially available ABC kit. We find that the method, which apparently relies on recognition of avidin's excess biotin binding sites by the biotin- peroxidase conjugate, is both faster and less labor intensive than photo- oxidation procedures in common use. The technique is readily adaptable to immunocytochemistry with biotinylated probes, as we demonstrate using anti- serotonin as an example.

Original languageEnglish (US)
Pages (from-to)263-273
Number of pages11
JournalJournal of Histochemistry and Cytochemistry
Volume46
Issue number2
StatePublished - Feb 1998

Fingerprint

Lasers
Coloring Agents
Avidin
Electrons
Biotin
Neurons
Confocal Microscopy
Fluorescent Dyes
Peroxidase
Serotonin
Electron Microscopy
Immunohistochemistry
Binding Sites
biocytin
neurobiotin

Keywords

  • Biocytin
  • Confocal microscopy
  • Manduca sexta
  • Musca domestica
  • Neurobiotin
  • Olfaction
  • Reconstruction
  • Synapse
  • Three-dimensional
  • Ultrastructure

ASJC Scopus subject areas

  • Anatomy
  • Cell Biology

Cite this

@article{5ae280a6b84f43f8b3d3a8ac0bec24b2,
title = "A rapid method for combined laser scanning confocal microscopic and electron microscopic visualization of biocytin or Neurobiotin-labeled neurons",
abstract = "Intracellular recording and dye filling are widely used to correlate the morphology of a neuron with its physiology. With laser scanning confocal microscopy, the complex shapes of labeled neurons in three dimensions can be reconstructed rapidly, but this requires fluorescent dyes. These dyes are neither permanent nor electron dense and therefore do not allow investigation by electron microscopy. Here we report a technique that quickly and easily converts a fluorescent label into a more stable and electron-dense stain. With this technique, a neuron is filled with Neurobiotin or biocytin, reacted with fluorophore-conjugated avidin, and imaged by confocal microscopy. To permit long-term storage or EM study, the fluorescent label is then converted to a stable electron-dense material by a single-step conversion using a commercially available ABC kit. We find that the method, which apparently relies on recognition of avidin's excess biotin binding sites by the biotin- peroxidase conjugate, is both faster and less labor intensive than photo- oxidation procedures in common use. The technique is readily adaptable to immunocytochemistry with biotinylated probes, as we demonstrate using anti- serotonin as an example.",
keywords = "Biocytin, Confocal microscopy, Manduca sexta, Musca domestica, Neurobiotin, Olfaction, Reconstruction, Synapse, Three-dimensional, Ultrastructure",
author = "Sun, {Xue J.} and Tolbert, {Leslie P} and Hildebrand, {John G} and Meinertzhagen, {Ian A.}",
year = "1998",
month = "2",
language = "English (US)",
volume = "46",
pages = "263--273",
journal = "Journal of Histochemistry and Cytochemistry",
issn = "0022-1554",
publisher = "Histochemical Society Inc.",
number = "2",

}

TY - JOUR

T1 - A rapid method for combined laser scanning confocal microscopic and electron microscopic visualization of biocytin or Neurobiotin-labeled neurons

AU - Sun, Xue J.

AU - Tolbert, Leslie P

AU - Hildebrand, John G

AU - Meinertzhagen, Ian A.

PY - 1998/2

Y1 - 1998/2

N2 - Intracellular recording and dye filling are widely used to correlate the morphology of a neuron with its physiology. With laser scanning confocal microscopy, the complex shapes of labeled neurons in three dimensions can be reconstructed rapidly, but this requires fluorescent dyes. These dyes are neither permanent nor electron dense and therefore do not allow investigation by electron microscopy. Here we report a technique that quickly and easily converts a fluorescent label into a more stable and electron-dense stain. With this technique, a neuron is filled with Neurobiotin or biocytin, reacted with fluorophore-conjugated avidin, and imaged by confocal microscopy. To permit long-term storage or EM study, the fluorescent label is then converted to a stable electron-dense material by a single-step conversion using a commercially available ABC kit. We find that the method, which apparently relies on recognition of avidin's excess biotin binding sites by the biotin- peroxidase conjugate, is both faster and less labor intensive than photo- oxidation procedures in common use. The technique is readily adaptable to immunocytochemistry with biotinylated probes, as we demonstrate using anti- serotonin as an example.

AB - Intracellular recording and dye filling are widely used to correlate the morphology of a neuron with its physiology. With laser scanning confocal microscopy, the complex shapes of labeled neurons in three dimensions can be reconstructed rapidly, but this requires fluorescent dyes. These dyes are neither permanent nor electron dense and therefore do not allow investigation by electron microscopy. Here we report a technique that quickly and easily converts a fluorescent label into a more stable and electron-dense stain. With this technique, a neuron is filled with Neurobiotin or biocytin, reacted with fluorophore-conjugated avidin, and imaged by confocal microscopy. To permit long-term storage or EM study, the fluorescent label is then converted to a stable electron-dense material by a single-step conversion using a commercially available ABC kit. We find that the method, which apparently relies on recognition of avidin's excess biotin binding sites by the biotin- peroxidase conjugate, is both faster and less labor intensive than photo- oxidation procedures in common use. The technique is readily adaptable to immunocytochemistry with biotinylated probes, as we demonstrate using anti- serotonin as an example.

KW - Biocytin

KW - Confocal microscopy

KW - Manduca sexta

KW - Musca domestica

KW - Neurobiotin

KW - Olfaction

KW - Reconstruction

KW - Synapse

KW - Three-dimensional

KW - Ultrastructure

UR - http://www.scopus.com/inward/record.url?scp=0031911560&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031911560&partnerID=8YFLogxK

M3 - Article

C2 - 9446834

AN - SCOPUS:0031911560

VL - 46

SP - 263

EP - 273

JO - Journal of Histochemistry and Cytochemistry

JF - Journal of Histochemistry and Cytochemistry

SN - 0022-1554

IS - 2

ER -