Horseradish peroxidase cDNA as a marker for electron microscopy in neurons

J Neurosci Methods. 2007 Sep 30;165(2):210-5. doi: 10.1016/j.jneumeth.2007.06.004. Epub 2007 Jun 12.

Abstract

A reliable and convenient marker of gene transfer in neurons is lacking for electron microscopy. To facilitate the use of molecular genetic approaches in such studies, we introduce the use of horseradish peroxidase (HRP) cDNA as a marker that identifies transfected neurons for correlated light and electron microscopy (EM). By flanking the cDNA with an endoplasmic reticulum (ER)-retention signal, expressed HRP was localized and retained inside the ER of neurons. The construct was placed under the control of the synapsin promoter resulting in neuron-specific expression of HRP. After transfection of hippocampal neurons in culture and visualization of HRP with diaminobenzidine (DAB) after fixation, both cell bodies and proximal dendrites of HRP-positive neurons were readily identifiable by light microscopy. After processing for EM, the heavily DAB-labeled ER was obvious in both cell bodies and dendrites of transfected neurons. In addition, the labeling of distal dendrites, axons, and synapses was apparent. The structural preservation of transfected neurons was optimal as is typical for glutaraldehyde-fixed tissue. Due to the ER-retention signal, HRP was contained inside the ER and the ultrastructure of all other components was not occluded and was quantitatively accessible. These characteristics make HRP co-transfection a potentially powerful tool for modern electron microscopy in both cell biology and neuroscience.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3,3'-Diaminobenzidine
  • Animals
  • Animals, Newborn
  • Biomarkers / analysis
  • Biomarkers / metabolism
  • Cells, Cultured
  • DNA, Complementary / analysis*
  • DNA, Complementary / genetics
  • DNA, Complementary / metabolism
  • Endoplasmic Reticulum / genetics
  • Genes, Reporter / genetics
  • Horseradish Peroxidase / genetics
  • Humans
  • Microscopy, Electron, Transmission / methods*
  • Molecular Biology / methods*
  • Neurons / metabolism*
  • Neurons / ultrastructure
  • Promoter Regions, Genetic / genetics
  • Rats
  • Staining and Labeling / methods*
  • Synapsins / genetics
  • Transfection / methods*

Substances

  • Biomarkers
  • DNA, Complementary
  • Synapsins
  • 3,3'-Diaminobenzidine
  • Horseradish Peroxidase