PMID: 11897811Mar 19, 2002Paper

Triple immunofluorescence staining with antibodies raised in the same species to study the complex innervation pattern of intrapulmonary chemoreceptors

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society
Inge BrounsDirk Adriaensen

Abstract

A general problem in immunocytochemistry is the development of a reliable multiple immunolabeling method when primary antibodies must be used that originate in the same species. We have developed a protocol for the immunodetection of three antigens in a single tissue preparation, using unconjugated primary antibodies raised in the same species. Immunocytochemical detection of neuronal nitric oxide synthase, calcitonin gene-related peptide, and calbindin D28k in the lung of rats demonstrated that part of the pulmonary neuroepithelial bodies are selectively contacted by at least three different nerve fiber populations. The first antigen was detected using tyramide signal amplification, a very sensitive method allowing a dilution of the first primary antibody far beyond the detection limit of fluorescently labeled secondary antibodies. The second antigen was visualized by a fluorophore-conjugated secondary monovalent Fab antibody that at the same time blocks the access of the third secondary antibody to the second primary antibody. Moreover, the monovalence of the Fab fragment prevents the third primary antibody from binding with the second-step secondary antibody. The triple staining technique described here is generally applicab...Continue Reading

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Citations

Dec 21, 2002·The Anatomical Record. Part A, Discoveries in Molecular, Cellular, and Evolutionary Biology·Paul J KempChris Peers
Aug 11, 2007·Histochemistry and Cell Biology·Ian De ProostDirk Adriaensen
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