title: Consequences of the bleed-through phenomenon in immunofluorescence of proteins forming radiation-induced nuclear foci.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Inserm U647, European Synchrotron Radiation Facility, Grenoble, France.
authors.forename: Wendy
authors.initials: W
authors.lastname: Rénier
authors.forename: Aurélie
authors.initials: A
authors.lastname: Joubert
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Bencokova
authors.forename: Jérôme
authors.initials: J
authors.lastname: Gastaldo
authors.forename: Catherine
authors.initials: C
authors.lastname: Massart
authors.forename: Nicolas
authors.initials: N
authors.lastname: Foray
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism / Q000473:pathology / Q000528:radiation effects*; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D005455:Fluorescent Antibody Technique / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D000076228:MRE11 Homologue Protein; D010766:Phosphorylation; D011839:Radiation, Ionizing; D012235:Rhodamines / Q000737:chemistry
chemical_list: D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; C468783:H2AX protein, human; D006657:Histones; C121766:MRE11 protein, human; D012235:Rhodamines; C009434:tetramethylrhodamine isothiocyanate; D000076228:MRE11 Homologue Protein; D016650:Fluorescein-5-isothiocyanate