Fixative composition alters distributions of immunoreactivity for glutaminase and two markers of nociceptive neurons, Nav1.8 and TRPV1, in the rat dorsal root ganglion.
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(v)1.8), 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 Na(v)1.8 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 Na(v)1.8, 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.
Hoffman
E Matthew
EM
Department of Anatomy and Cell Biology, Oklahoma State University Center for Health Sciences, Tulsa, Oklahoma 74107, USA.
Schechter
Ruben
R
Miller
Kenneth E
KE
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D005404:Fixatives; D005455:Fluorescent Antibody Technique; D005557:Formaldehyde; D005727:Ganglia, Spinal / Q000378:metabolism*; D005972:Glutaminase / Q000378:metabolism*; D007106:Immune Sera; D062559:NAV1.8 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins / Q000096:biosynthesis*; D009619:Nociceptors / Q000378:metabolism*; D010851:Picrates; D051381:Rats; D017207:Rats, Sprague-Dawley; D015222:Sodium Channels / Q000096:biosynthesis*; D050916:TRPV Cation Channels / Q000096:biosynthesis*; D013997:Time Factors; D016707:Tissue Fixation
D015415:Biomarkers; D005404:Fixatives; D007106:Immune Sera; D062559:NAV1.8 Voltage-Gated Sodium Channel; D009419:Nerve Tissue Proteins; D010851:Picrates; C568319:Scn10a protein, rat; D015222:Sodium Channels; D050916:TRPV Cation Channels; C493922:Trpv1 protein, rat; D005557:Formaldehyde; C005858:picric acid; D005972:Glutaminase