Immunohistochemical localization of choline acetyltransferase using a monoclonal antibody: a radioautographic method.
Monoclonal antibodies to rat striatal choline acetyltransferase were produced by fusion of sensitized mouse lymphocytes with murine plasmacytoma (NS1) cells. Two stable anti-choline acetyltransferase lines were established by limiting dilution cloning. Specificity of antibody was established by the following criteria: (1) on an enzyme linked immunosorbant assay, antibodies reacted against choline acetyltransferase which was highly purified; (2) by immunoprecipitation, monoclonal antibody bound to its antigen and precipitated choline acetyltransferase activity from solution, when used in conjunction with rabbit antimouse IgG; and (3) monoclonal antibody was shown to specifically localize cholinergic neurons. The monoclonal antibody to choline acetyltransferase was radiolabeled in culture by incubating hybridomas in medium containing 3H-labeled amino acids. This 3H-labeled antibody was used for radioautography on cryostat sections of rat peripheral and central nervous systems. In a sampling of areas, highly specific labeling of cholinergic structures was afforded at both light and electron microscopic levels. Double labeling of tyrosine hydroxylase, a catecholaminergic marker, and choline acetyltransferase was carried out by reacting sections first with the 3H-labeled antibody to choline acetyltransferase and then with rabbit antibody to tyrosine hydroxylase. The choline acetyltransferase label was radioautographically processed and tyrosine hydroxylase was visualized by the peroxidase-antiperoxidase method. The combined techniques of peroxidase and radioautographic histochemistry provide permanent electron dense labels which can be examined simultaneously within a single histologic section.
D000818:Animals; D000906:Antibodies / Q000032:analysis; D000911:Antibodies, Monoclonal / Q000032:analysis; D001345:Autoradiography; D002795:Choline O-Acetyltransferase / Q000032:analysis* / Q000276:immunology; D006651:Histocytochemistry; D007124:Immunoenzyme Techniques; D007158:Immunologic Techniques; D008722:Methods; D051379:Mice; D008815:Mice, Inbred Strains; D008854:Microscopy, Electron; D051381:Rats; D014446:Tyrosine 3-Monooxygenase / Q000276:immunology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D014446:Tyrosine 3-Monooxygenase; D002795:Choline O-Acetyltransferase