title: Triple immunofluorescence staining with antibodies raised in the same species to study the complex innervation pattern of intrapulmonary chemoreceptors.
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 applicable, uses commercially available products only, and allows the detection of three antigens in the same preparation with primary antibodies that are raised in the same species.
authors.affiliation: Laboratory of Cell Biology and Histology, University of Antwerp (RUCA), Groenenborgerlaan 171, B-2020 Antwerp, Belgium.
authors.forename: Inge
authors.initials: I
authors.lastname: Brouns
authors.forename: Luc
authors.initials: L
authors.lastname: Van Nassauw
authors.forename: Jeroen
authors.initials: J
authors.lastname: Van Genechten
authors.forename: Mariusz
authors.initials: M
authors.lastname: Majewski
authors.forename: Dietrich W
authors.initials: DW
authors.lastname: Scheuermann
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Timmermans
authors.forename: Dirk
authors.initials: D
authors.lastname: Adriaensen
mesh_terms: D000818:Animals; D064092:Calbindin 1; D064026:Calbindins; D015740:Calcitonin Gene-Related Peptide / Q000276:immunology; D002628:Chemoreceptor Cells / Q000276:immunology*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D007518:Isoantibodies; D008168:Lung / Q000294:innervation; D019001:Nitric Oxide Synthase / Q000276:immunology; D052248:Nitric Oxide Synthase Type I; D051381:Rats; D017208:Rats, Wistar; D064030:S100 Calcium Binding Protein G / Q000276:immunology
chemical_list: C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D007518:Isoantibodies; D064030:S100 Calcium Binding Protein G; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D015740:Calcitonin Gene-Related Peptide