1. Consequences of the bleed-through phenomenon in immunofluorescence of proteins forming radiation-induced nuclear foci
- Author
-
Zuzana Bencokova, Catherine Massart, Wendy Rénier, Jérôme Gastaldo, Nicolas Foray, Aurélie Joubert, European Synchrotron Radiation Facility (ESRF), Rayonnement Synchrotron et Recherche Medicale (RSRM), Université Joseph Fourier - Grenoble 1 (UJF)-European Synchrotron Radiation Facility (ESRF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Radioprotection et de Sureté Nucléaire, Institut de Radioprotection et de Sûreté Nucléaire (IRSN), Serduc, Raphael, and European Synchrotron Radiation Facility (ESRF)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)
- Subjects
DNA Repair ,Fluorescent Antibody Technique ,Ionizing radiation ,Histones ,chemistry.chemical_compound ,0302 clinical medicine ,Radiation, Ionizing ,Phosphorylation ,Fluorescein isothiocyanate ,MESH: Fluorescent Antibody Technique ,MESH: DNA Repair ,MESH: Histones ,0303 health sciences ,MRE11 Homologue Protein ,Radiological and Ultrasound Technology ,medicine.diagnostic_test ,MESH: Kinetics ,MESH: Fluorescent Dyes ,Fluorescence ,3. Good health ,DNA-Binding Proteins ,030220 oncology & carcinogenesis ,Fluorescein-5-isothiocyanate ,MESH: Cell Nucleus ,DNA damage ,DNA repair ,Biology ,MESH: Radiation, Ionizing ,MESH: Rhodamines ,Immunofluorescence ,03 medical and health sciences ,MESH: Fluorescein-5-isothiocyanate ,medicine ,Humans ,Radiology, Nuclear Medicine and imaging ,030304 developmental biology ,Fluorescent Dyes ,MESH: DNA Damage ,Cell Nucleus ,MESH: Humans ,MESH: Phosphorylation ,Rhodamines ,Molecular biology ,Hsp70 ,Kinetics ,chemistry ,Biophysics ,MESH: DNA-Binding Proteins ,DNA Damage - Abstract
International audience; PURPOSE: By allowing the visualization of the proteins inside cells, the immunofluorescence technique has revolutionized our view of events that follow radiation response. Particularly, the formation of nuclear foci, their kinetic of appearance and disappearance, and the association-dissociation of protein partners are useful endpoints to better understand the effects of ionizing radiation. Recently, the technique based on the phosphorylation of the histone 2A family, member X (H2AX) has generated a plethora of reports concerning the interaction between the major proteins involved in DNA repair and stress signaling pathways. However, some unavoidable overlaps of excitation and emission wavelength spectra (the so-called bleed-through phenomenon) of the available fluorescent markers are still generating discrepancies and misinterpretations in the choreography of DNA damage response. Biases are particularly strong with the fluorescein isothiocyanate (FITC)-rhodamine couple, tetramethyl rhodamine iso-thiocyanate (TRITC), the most extensively used markers. METHOD AND RESULTS: Here, two representative examples of biased co-immunofluorescence with pH2AX proteins that form radiation-induced nuclear foci or not are presented. A brief review of literature points out differences in kinetic of appearance and association-dissociation of radiation-induced pH2AX and MRE11 foci. CONCLUSION: Through this report, we would like authors to consider more carefully protein co-localizations by performing systematically, before any co-immunofluorescence, immunofluorescence of each protein separately to avoid bleed-through artifacts.
- Published
- 2007
- Full Text
- View/download PDF