Radioimmunochemical methods for the quantitative autoradiographic determination of antigens in brain and other tissues.
1. We have used horseradish peroxidase-conjugated protein A- and 125I-protein A to develop immunohistochemical and radioimmunohistochemical methods for the localization of antigens in brain and other tissues of the rat. 2. We visualized methionine-enkephalin fibers in the rat brain by incubating tissue sections with a specific polyclonal antibody and peroxidase-conjugated protein A. The method is simple, fast, and less expensive and more sensitive than classical immunohistochemical techniques and the principle could be used to visualize many other tissue antigens. 3. Incubation of tissue samples with specific polyclonal antibodies and 125I-protein A, followed by autoradiography, allows the permanent recording of the radioimmunohistochemical localization of brain methionine-enkephalin, tyrosine hydroxylase, and angiotensin-converting enzyme and of pituitary vasopressin and could be applied to the localization of many other tissue antigens. 4. A new quantitative radioimmunohistochemical technique for methionine-enkephalin allows the determination of the endogenous peptide content in discrete brain nuclei from 16-microns-thick sections. The method is based on the quantitative determination of the amount of 125I-protein A bound to specific tissue areas after incubation with a specific polyclonal antibody, followed by autoradiography and computerized microdensitometry. To quantify the endogenous peptide content, the values obtained are interpolated into a methionine-enkephalin internal standard curve. This standard curve was constructed by measuring endogenous concentrations of methionine-enkephalin by radioimmunoassay in specific brain regions and correlating these values with quantitative autoradiographic determinations in homologous areas of adjacent sections. Similar methods can be developed for other tissue antigens. 5. These new methods allow for the localization and quantification of tissue antigens in very discrete areas of the brain and other tissues and have a wide application in neurobiology and pathology.
Correa
F M
FM
Unit on Preclinical Neuropharmacology, National Institute of Mental Health, Bethesda, Maryland 20892.
Guilhaume
S S
SS
Saavedra
J M
JM
D000818:Animals; D001345:Autoradiography / Q000379:methods*; D001921:Brain / Q000378:metabolism*; D004744:Enkephalin, Methionine / Q000032:analysis*; D006735:Horseradish Peroxidase; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013205:Staphylococcal Protein A
D013205:Staphylococcal Protein A; D004744:Enkephalin, Methionine; D006735:Horseradish Peroxidase