Method Article

Immunofluorescence Microscopy for the Analysis of DNA Double-Strand Breaks

July 8th, 2025

In This Article

Abstract

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Source: Popp, H. D., et al. Immunofluorescence Microscopy of γH2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks. J. Vis. Exp. (2017)

In this video, we describe the immunofluorescence microscopy technique to detect specific DNA damage response proteins localized at the sites of DNA double-strand breaks in human mononuclear cells. The proteins are recognized by specific primary antibodies, which in turn bind to fluorophore-tagged secondary antibodies that fluoresce during microscopic visualization, enabling visualization of the proteins as distinct fluorescent foci within the cell nuclei.

Protocol

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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Preparation of the cytospins

  1. Prepare two cytospins with mononuclear cells of the patient samples (i.e., one cytospin for γH2AX immunofluorescence staining and one cytospin for γH2AX and 53BP1 combined immunofluorescence staining) by centrifugation (300 x g, 10 min) of 1.0 x 105 cells for each preparation (Figure 1 and 2

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Results

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Spectroscopy components setup for optical excitation and analysis, including cuvettes and holders.
Figure 1: Device for preparing the cytospins.

Cytospin immunostaining, γH2AX/53BP1, slide diagram, protein analysis.
Figure 2.......

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Disclosures

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No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Diagnostic microscope slidesThermo ScientificER-203B-CE24Microscope slides
Megafuge 1.0 RHeraeus75003060Tabletop centrifuge
Cytospin device
LidHeraeus76003422Lid for working without micro-tubes
Cyto containerHeraeus75003416Cyto container with 2 conical bores
Clip carrierHeraeus75003414Carrier for holding a cyto container and a slide
Support insertHeraeus75003417Support insert for holding a clip carrier
ParaformaldehydeSigma-AldrichP6148Fixation agent
Phosphate-buffered salineSigma-AldrichD8537Balanced salt solution
ChemiblockerMerck Millipore2170Blocking agent
Mouse monoclonal anti-γH2AX antibody (JBW301)Merck Millipore05-636Primary antibody for detection of γH2AX
Polyclonal rabbit anti-53BP1 antibody (NB100-304)Novus BiologicalsNB100-304Primary antibody for detection of 53BP1
Alexa Fluor 488-conjugated goat anti-mouse antibodyInvitrogenA-11001Secondary antibody
Alexa Fluor 555-conjugated donkey anti-rabbit antibodyInvitrogenA-31572 Secondary antibody
Vectashield mounting mediumVector LaboratoriesH-1200Contains DAPI for staining of DNA
Axio Scope.A1Zeiss490035Fluorescence microscope
Cool Cube 1 CCD cameraMetasystemsH-0310-010-MSCamera system for digital recording
Isis softwareMetasystemsNot applicableMicroscope software

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Tags

Immunofluorescence MicroscopyDNA Double Strand BreaksGamma H2AX53BP1Fluorescence MicroscopyPrimary AntibodiesSecondary AntibodiesDAPI StainingCell PermeabilizationFluorescent Foci

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