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PMID: 2875047 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

A combined light and electron microscopic immunocytochemical method for the simultaneous localization of multiple tissue antigens. Tyrosine hydroxylase immunoreactive innervation of corticotropin releasing factor synthesizing neurons in the paraventricular nucleus of the rat.

Histochemistry ·Vol. 85 ·No. 2 ·1986-00-00 ·Pages 95-106

Liposits Z, Sherman D, Phelix C, Paull WK

Abstract

In order to study the morphological interrelationships between immunocytochemically identified neuronal systems, a double labelling procedure - suitable for correlative light and electron microscopic observations - is introduced. The technique is based on the consecutive use of the silver-gold (SG) intensified and non-intensified forms of the oxidized 3,3'-diaminobenzidine (DAB) chromogen in the framework of the peroxidase-antiperoxidase complex (PAP) indirect immunocytochemical procedure. The first tissue antigen is detected by the SG intensified DAB chromogen, which has a black color and high electron density. The structures containing the second antigen are visualized by the non-intensified DAB-endproduct, which is less electron-dense than the silver-gold amplified form and is brown. The metallic shield that forms around the labeled antibody sequences associated with the first antigen prevents non-specific binding of immunoglobulins used for the detection of the second tissue antigen. The application of this method for the simultaneous detection of tyrosine hydroxylase (TH)- and corticotropin releasing factor (CRF)-immunoreactive structures revealed that black colored TH-immunopositive fibers contacted brown colored CRF-synthesizing neurons in the hypothalamic paraventricular nucleus. The juxtaposition of TH- and CRF-containing elements was apparent in both thick vibratome (40 micron) and semithin (1 micron) sections. At the ultrastructural level, TH-positive terminals - labeled by silver-gold grains - were observed to establish asymmetric synapses with both CRF- and TH-immunoreactive neurons. The former finding indicates a direct, TH-immunopositive, catecholaminergic influence upon the hypothalamic CRF system, while the latter demonstrates the existence of intrinsic connections between TH-positive elements.

MeSH Terms
Animals Corticotropin-Releasing Hormone/metabolism Gold Immunochemistry/methods Microscopy, Electron/methods Paraventricular Hypothalamic Nucleus/metabolism,ultrastructure Rats Tyrosine 3-Monooxygenase/metabolism
Chemicals
Gold Corticotropin-Releasing Hormone Tyrosine 3-Monooxygenase
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Liposits Z
Sherman D
Phelix C
Paull W K
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51 references, click to expand
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Article Info
Journal
Histochemistry
Abbr.
Histochemistry
ISSN
0301-5564
DOI
10.1007/BF00491754
Published
1986-00-00
Pages
95-106
Language
English
Region
Germany
NLM ID
0411300
Subset
IM
Grants
NINDS NIH HHS · NS19266 · United States
External Links
PubMed source DOI: 10.1007/BF00491754
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