Electron microscopy of intracellularly labeled neurons in the hippocampal slice preparation

D. D. Kunkel, H. E. Scharfman, D. L. Schmiege, Philip A Schwartzkroin

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

We have assessed the properties of three intracellular markers, horseradish peroxidase, biocytin/Neurobiotin, and Lucifer Yellow, and have compared their usefulness as neuronal markers for light and electron microscopic visualization. Neurons in the acute slice preparation of rat hippocampus were filled with one of these markers, and the marker was converted to an optical and electron-dense reaction product. Dimethylsulfoxide (DMSO) greatly facilitated penetration of recognition reagents while preserving membrane integrity. The markers were compared with respect to injection parameters, mobility and recognition, stability and visibility, and ultrastructural clarity. Horseradish peroxidase (HRP)- labeled neurons, recognized histochemically with diaminobenzedine (DAB), were easily visualized by the density of the DAB reaction product; however, the electron density was often so great as to obscure ultrastructural details. Biocytin (BC)-/Neurobiotin (NB)-labeled neurons were recognized by avidin- HRP, followed by histochemical localization of HRP with DAB. The optically dense reaction product gave complete visualization of the soma and processes at the light microscopic level. The electron density was homogeneously distributed throughout the cell, so that ultrastructural features were easily identified. Lucifer Yellow (LY), a fluorescent marker, was converted to an optical and electron-dense reaction product via immunocytochemical staining with a rabbit anti-LY antibody, followed by goat anti-rabbit IgG-HRP and DAB histochemical localization. Similar to BC/NB, the reaction product was evenly dispersed, providing good light microscopic and ultrastructural clarity. Under our experimental conditions, BC/NB and LY were superior markers that could be used routinely to label neurons, and give excellent visualization not only at the light but also at the electron microscopic level.

Original languageEnglish (US)
Pages (from-to)67-84
Number of pages18
JournalMicroscopy Research and Technique
Volume24
Issue number1
DOIs
StatePublished - 1993
Externally publishedYes

Keywords

  • Biocytin
  • Horseradish peroxidase
  • Intracellular labeling
  • Lucifer Yellow
  • Neurobiotin
  • Ultrastructure

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Anatomy
  • Instrumentation

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