Neurochemical identification of fos-positive neurons using two-colour immunoperoxidase staining

Smith D.W. and Day T.A. (1993) Neurochemical identification of fos-positive neurons using two-colour immunoperoxidase staining. Journal of Neuroscience Methods, 47 1-2: 73-83. doi:10.1016/0165-0270(93)90023-K


Author Smith D.W.
Day T.A.
Title Neurochemical identification of fos-positive neurons using two-colour immunoperoxidase staining
Journal name Journal of Neuroscience Methods   Check publisher's open access policy
ISSN 0165-0270
Publication date 1993-01-01
Sub-type Article (original research)
DOI 10.1016/0165-0270(93)90023-K
Volume 47
Issue 1-2
Start page 73
End page 83
Total pages 11
Subject 2800 Neuroscience
Abstract The discovery of immediate early genes (IEG) has provided neuroscientists with a new functional mapping technique. Labelling of neural tissue for the protein product of IEG provides an activity map with single-cell resolution. When combined with labelling for the chemical identity of the neuron, this provides a powerful tool for the investigation of specific cell populations along a neuraxis. Here we describe in detail a method which allows simultaneous bright-field visualization of neurochemically identified cells displaying increased IEG expression. This technique is evaluated in tissue from rats subjected to stimuli known to induce the expression of the IEG c-fos in various medullary catecholaminergic and hypothalamic neurosecretory cell groups. A 2-colour immunoperoxidase technique was used to visualize Fos, the nuclear protein product of c-fos, and the cytoplasmic antigens tyrosine hydroxylase (TH), phenylethanolamine N-methyl transferase (PNMT), oxytocin (OT) and vasopressin (VP). This involved simultaneous application of primary antibodies raised in different species followed by sequential application of appropriate biotinylated secondary antibodies and the avidin-biotin-peroxidase technique. Fos was visualized with nickel-intensified diaminobenzidine (Ni-DAB) in the first sequence while TH, PNMT, OT or VP were visualized with DAB alone, resulting in readily distinguishable black and amber reaction products, respectively. This dual immunoperoxidase technique is time saving compared to techniques using sequential application of primary antibodies and avoids the disadvantages associated with fluorescence techniques.
Keyword Dual immunoperoxidase staining
Fos
Functional mapping
Immediate early gene
Oxytocin
Phenylethanolamine N-methyl transferase
Tyrosine hydroxylase
Vasopressin
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collection: Scopus Import - Archived
 
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