Open AccessAn indirect immunofluorescence double staining procedure for the simultaneous flow cytometric measurement of iodo‐ and chlorodeoxyuridine incorporated into DNA
Author(s) -
Bakker P. J. M.,
Stap J.,
Tukker C. J.,
van Oven C. H.,
Veenhof C. H. N.,
Aten J.
Publication year - 1991
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.990120412
Subject(s) - immunofluorescence , monoclonal antibody , staining , flow cytometry , antibody , microbiology and biotechnology , primary and secondary antibodies , chemistry , conjugated system , tris , bromodeoxyuridine , indirect immunofluorescence , fluorescence , dna , biology , chromatography , biochemistry , immunology , cell growth , genetics , polymer , physics , organic chemistry , quantum mechanics
In this paper we describe an indirect fluorescence double staining procedure for the simultaneous detection of IdUrd and CldUrd in the same cell nucleus. Two commercially available antibodies were selected for this purpose. A rat antiBrdUrd monoclonal antibody from Seralab was found to bind specifically to CldUrd and BrdUrd. A mouse monoclonal anti‐BrdUrd antibody from Becton Dickinson used in a 1:2 dilution binds to all halogenated deoxyuridines but, when the cells were extensively washed with Tris buffer with a high salt concentration, almost no binding to CldUrd was observed. An immunofluorescence procedure was developed, based on these primary antibodies, raised in different species (rat and mouse), in combination with highly purified second antibodies: FITC conjugated goat antirat and Texas‐Red conjugated goat antimouse.