Fixative composition alters distributions of immunoreactivity for glutaminase and two markers of nociceptive neurons, Nav1.8 and TRPV1, in the rat dorsal root ganglion

E. Matthew Hoffman, Ruben Schechter, Kenneth Miller

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20 Citations (Scopus)

Abstract

Most, if not all, dorsal root ganglion (DRG) neurons use the neurotransmitter glutamate. There are, however, conflicting reports of the percentages of DRG neurons that express glutaminase (GLS), the enzyme that synthesizes glutamate, ranging from 30% to 100% of DRG neurons. Defining DRG neuron populations by the expression of proteins like GLS, which indicates function, is routinely accomplished with immunolabeling techniques. Proper characterization of DRG neuron populations relies on accurate detection of such antigens. It is known intuitively that fixation can alter immunoreactivity (IR). In this study, we compared the effects of five formaldehyde concentrations between 0.25% and 4.0% (w/v) and five picric acid concentrations between 0.0% and 0.8% (w/v) on the IR of GLS, the voltage-gated sodium channel 1.8 (Na v1.8), and the capsaicin receptor TRPV1. We also compared the effects of five incubation time lengths from 2 to 192 hr, in primary antiserum on IR. Lowering formaldehyde concentration elevated IR for all three antigens, while raising picric acid concentration increased Nav1.8 and TRPV1 IR. Increasing IR improved detection sensitivity, which led to higher percentages of labeled DRG neurons. By selecting fixation conditions that optimized IR, we found that all DRG neurons express GLS, 69% of neurons express Nav1.8, and 77% of neurons express TRPV1, indicating that some previous studies may have underestimated the percentages of DRG neurons expressing these proteins. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Original languageEnglish
Pages (from-to)329-344
Number of pages16
JournalJournal of Histochemistry and Cytochemistry
Volume58
Issue number4
DOIs
StatePublished - 1 Apr 2010

Fingerprint

Glutaminase
Fixatives
Nociceptors
Spinal Ganglia
Neurons
Formaldehyde
Glutamic Acid
Voltage-Gated Sodium Channels
TRPV Cation Channels
Antigens
Population
Neurotransmitter Agents
Immune Sera
Proteins
Immunohistochemistry

Keywords

  • Glutamate
  • Image analysis
  • ImageJ
  • Immunohistochemistry
  • Paraformaldehyde
  • Phosphate-activated glutaminase
  • Primary afferent neurons

Cite this

@article{967f4b4bc8034fd3998d4d4282408bd2,
title = "Fixative composition alters distributions of immunoreactivity for glutaminase and two markers of nociceptive neurons, Nav1.8 and TRPV1, in the rat dorsal root ganglion",
abstract = "Most, if not all, dorsal root ganglion (DRG) neurons use the neurotransmitter glutamate. There are, however, conflicting reports of the percentages of DRG neurons that express glutaminase (GLS), the enzyme that synthesizes glutamate, ranging from 30{\%} to 100{\%} of DRG neurons. Defining DRG neuron populations by the expression of proteins like GLS, which indicates function, is routinely accomplished with immunolabeling techniques. Proper characterization of DRG neuron populations relies on accurate detection of such antigens. It is known intuitively that fixation can alter immunoreactivity (IR). In this study, we compared the effects of five formaldehyde concentrations between 0.25{\%} and 4.0{\%} (w/v) and five picric acid concentrations between 0.0{\%} and 0.8{\%} (w/v) on the IR of GLS, the voltage-gated sodium channel 1.8 (Na v1.8), and the capsaicin receptor TRPV1. We also compared the effects of five incubation time lengths from 2 to 192 hr, in primary antiserum on IR. Lowering formaldehyde concentration elevated IR for all three antigens, while raising picric acid concentration increased Nav1.8 and TRPV1 IR. Increasing IR improved detection sensitivity, which led to higher percentages of labeled DRG neurons. By selecting fixation conditions that optimized IR, we found that all DRG neurons express GLS, 69{\%} of neurons express Nav1.8, and 77{\%} of neurons express TRPV1, indicating that some previous studies may have underestimated the percentages of DRG neurons expressing these proteins. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.",
keywords = "Glutamate, Image analysis, ImageJ, Immunohistochemistry, Paraformaldehyde, Phosphate-activated glutaminase, Primary afferent neurons",
author = "Hoffman, {E. Matthew} and Ruben Schechter and Kenneth Miller",
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TY - JOUR

T1 - Fixative composition alters distributions of immunoreactivity for glutaminase and two markers of nociceptive neurons, Nav1.8 and TRPV1, in the rat dorsal root ganglion

AU - Hoffman, E. Matthew

AU - Schechter, Ruben

AU - Miller, Kenneth

PY - 2010/4/1

Y1 - 2010/4/1

N2 - Most, if not all, dorsal root ganglion (DRG) neurons use the neurotransmitter glutamate. There are, however, conflicting reports of the percentages of DRG neurons that express glutaminase (GLS), the enzyme that synthesizes glutamate, ranging from 30% to 100% of DRG neurons. Defining DRG neuron populations by the expression of proteins like GLS, which indicates function, is routinely accomplished with immunolabeling techniques. Proper characterization of DRG neuron populations relies on accurate detection of such antigens. It is known intuitively that fixation can alter immunoreactivity (IR). In this study, we compared the effects of five formaldehyde concentrations between 0.25% and 4.0% (w/v) and five picric acid concentrations between 0.0% and 0.8% (w/v) on the IR of GLS, the voltage-gated sodium channel 1.8 (Na v1.8), and the capsaicin receptor TRPV1. We also compared the effects of five incubation time lengths from 2 to 192 hr, in primary antiserum on IR. Lowering formaldehyde concentration elevated IR for all three antigens, while raising picric acid concentration increased Nav1.8 and TRPV1 IR. Increasing IR improved detection sensitivity, which led to higher percentages of labeled DRG neurons. By selecting fixation conditions that optimized IR, we found that all DRG neurons express GLS, 69% of neurons express Nav1.8, and 77% of neurons express TRPV1, indicating that some previous studies may have underestimated the percentages of DRG neurons expressing these proteins. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

AB - Most, if not all, dorsal root ganglion (DRG) neurons use the neurotransmitter glutamate. There are, however, conflicting reports of the percentages of DRG neurons that express glutaminase (GLS), the enzyme that synthesizes glutamate, ranging from 30% to 100% of DRG neurons. Defining DRG neuron populations by the expression of proteins like GLS, which indicates function, is routinely accomplished with immunolabeling techniques. Proper characterization of DRG neuron populations relies on accurate detection of such antigens. It is known intuitively that fixation can alter immunoreactivity (IR). In this study, we compared the effects of five formaldehyde concentrations between 0.25% and 4.0% (w/v) and five picric acid concentrations between 0.0% and 0.8% (w/v) on the IR of GLS, the voltage-gated sodium channel 1.8 (Na v1.8), and the capsaicin receptor TRPV1. We also compared the effects of five incubation time lengths from 2 to 192 hr, in primary antiserum on IR. Lowering formaldehyde concentration elevated IR for all three antigens, while raising picric acid concentration increased Nav1.8 and TRPV1 IR. Increasing IR improved detection sensitivity, which led to higher percentages of labeled DRG neurons. By selecting fixation conditions that optimized IR, we found that all DRG neurons express GLS, 69% of neurons express Nav1.8, and 77% of neurons express TRPV1, indicating that some previous studies may have underestimated the percentages of DRG neurons expressing these proteins. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

KW - Glutamate

KW - Image analysis

KW - ImageJ

KW - Immunohistochemistry

KW - Paraformaldehyde

KW - Phosphate-activated glutaminase

KW - Primary afferent neurons

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U2 - 10.1369/jhc.2009.954008

DO - 10.1369/jhc.2009.954008

M3 - Article

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VL - 58

SP - 329

EP - 344

JO - Journal of Histochemistry and Cytochemistry

JF - Journal of Histochemistry and Cytochemistry

SN - 0022-1554

IS - 4

ER -