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Rapid immunofluorescent determination of cells in the S phase in pea root meristems: An alternative to autoradiography
Author(s) -
Levi Marisa,
Sparvoli Elio,
Sgorbati Sergio,
Chiatante Donato
Publication year - 1987
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1987.tb04618.x
Subject(s) - dapi , pisum , meristem , microbiology and biotechnology , bromodeoxyuridine , biology , population , nuclear dna , dna , sativum , fluorescein isothiocyanate , fluorescence , vicia faba , fluorescent labelling , chemistry , staining , biochemistry , botany , genetics , demography , physics , quantum mechanics , sociology , cell growth , mitochondrial dna , gene
Root meristems of Pisum sativum L. cv. Lincoln were pulsed with bromodeoxyuridine, and after partial DNA denaturation the incorporated precursor was detected with an indirect immunofluorescent method by means of commercial anti‐bromodeoxyuridine monoclonal antibody and fluorescein isothiocyanate‐labeled secondary antibody. The nuclei were counterstained with the DNA‐specific fluorochrome 4′, 6‐diamidino‐2‐phenylindole (DAPI). The labeling indices (percentage of labeled calls) determined on slides prepared with the same nuclear population were very similar and in strict accordance with autoradiographic data. When DAPI fluorescence of the nuclei was measured, the unlabeled nuclei coincided with the G1 and part of the G2 regions, whereas all the labeled nuclei were in the S or G2 region. The method is reliable and, in contrast to autoradiography, data can be collected rapidly and almost immediately after the pulse. Furthermore, labeling index and the distribution of DNA contents can be obtained from the same side.