Autoradiographic detection of (/sup 125/I)-secondary antiserum: a sensitive light and electron microscopic labeling method compatible with peroxidase immunocytochemistry for dual localization of neuronal antigens

Journal Article · Sun Jun 01 00:00:00 EDT 1986 · J. Histochem. Cytochem.; (United States)

DOI:https://doi.org/10.1177/34.6.2422251· OSTI ID:5528752

Pickel, V M; Chan, J; Milner, T A

We examined whether autoradiographic localization of (/sup 125/I)-antirabbit immunoglobulin (IgG) was suitable for light and electron microscopic detection of a rabbit antiserum to the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), and whether autoradiographic and peroxidase labeling could be combined for simultaneous immunocytochemical identification of TH and neuropeptides in brain. Adult rat brains were fixed by aortic arch perfusion with acrolein and paraformaldehyde. Vibratome sections of the fixed tissues were incubated with various dilutions of TH antiserum followed by (/sup 125/I)-secondary IgG. These sections were then directly processed for autoradiography or were incubated with rabbit antiserum to substance P (SP) or methionine (Met5)-enkephalin (ME). These latter sections were then processed by the peroxidase-antiperoxidase (PAP) or conjugated peroxidase methods followed by autoradiography. Exposure periods of 12-20 days for light microscopy or 90 days for electron microscopy yielded substantial accumulations of silver grains even at the highest (1:30,000) dilution of TH antiserum. At this dilution, immunoreactivity for TH was virtually nondetectable by PAP and conjugated peroxidase methods. The differential sensitivities of the autoradiographic versus peroxidase methods provided a means for separable identification of rabbit antiserum to TH and to SP or ME. Ultrastructural analysis of the catecholaminergic neurons in the medial nuclei of the solitary tract (NTS) showed selective cytoplasmic localization of silver grains for (/sup 125/I)-labeling of TH in perikarya, dendrites, and terminals. Within single thin sections prepared for dual labeling, the peroxidase marker for SP and for ME was differentially localized with respect to autoradiographic labeling of TH.

• Research Organization: Cornell Univ. Medical College, New York, NY
• OSTI ID: 5528752
• Journal Information: J. Histochem. Cytochem.; (United States), Journal Name: J. Histochem. Cytochem.; (United States) Vol. 6; ISSN JHCYA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANIMAL CELLS
ANIMALS
ANTIGENS
AUTONOMIC NERVOUS SYSTEM AGENTS
AUTORADIOGRAPHY
BIOLOGICAL LOCALIZATION
BODY
BRAIN
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL NUCLEI
CENTRAL NERVOUS SYSTEM
DRUGS
ENDORPHINS
ENKEPHALINS
ENZYMES
GLOBULINS
HYDROXYLASES
IMMUNOGLOBULINS
ISOENZYMES
LABELLING
LIPOTROPIC FACTORS
MAMMALS
METHIONINE
NERVE CELLS
NERVOUS SYSTEM
NEUROREGULATORS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
OXIDOREDUCTASES
PEPTIDES
PEROXIDASES
POLYPEPTIDES
PROTEINS
RABBITS
RATS
RODENTS
SOMATIC CELLS
VERTEBRATES