Dual immunocytochemistry using 125I-labeled protein A: a new electron microscopic technique applied to the investigation of chemical connectivity and axonal transmitter co-localization in the brain.
We have developed a double labeling immunocytochemical method utilizing peroxidase conjugated Fab fragments and 125I-labeled protein A to localize two neuronal markers on the same light or electron microscopic section with primary antibodies raised in the same animal species. The technique is applicable to the study of chemical connectivity in the brain, as illustrated by data obtained in the hypothalamus using rabbit polyclonal antisera against tyrosine hydroxylase (TH), phenylethanolamine-N-methyltransferase (PNMT), neuropeptide Y (NPY), and vasoactive intestinal peptide (VIP). Moreover, due to a high level of sensitivity and resolution, the technique offers considerable advantages over many previously developed dual labeling immunocytochemical methods for the demonstration of transmitter axonal co-localizations. Utilizing the peroxidase Fab/[125I]protein A method, we present here the first direct evidence that PNMT is present in many endings also containing NPY in the thalamic and hypothalamic paraventricular nuclei and in the arcuate nucleus. The method also may be combined as required with other labeling methods for localizing more than two neurochemical markers on one and the same electron microscopic section.
Kachidian
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P
Equipe de Neuromorphologie Fonctionnelle, Laboratoire de Neurobiologie, CNRS, Marseille, France.
Bosler
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O
D015100:3,3'-Diaminobenzidine; D000818:Animals; D001923:Brain Chemistry; D007031:Hypothalamus / Q000737:chemistry; D007106:Immune Sera; D007124:Immunoenzyme Techniques; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007457:Iodine Radioisotopes; D008297:Male; D016253:Microscopy, Immunoelectron / Q000379:methods*; D009478:Neuropeptide Y / Q000032:analysis* / Q000276:immunology; D010625:Phenylethanolamine N-Methyltransferase / Q000032:analysis* / Q000276:immunology; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D013205:Staphylococcal Protein A; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis* / Q000276:immunology; D014660:Vasoactive Intestinal Peptide / Q000032:analysis* / Q000276:immunology
D007106:Immune Sera; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D009478:Neuropeptide Y; D013205:Staphylococcal Protein A; D015100:3,3'-Diaminobenzidine; D014660:Vasoactive Intestinal Peptide; D014446:Tyrosine 3-Monooxygenase; D010625:Phenylethanolamine N-Methyltransferase