Immunofluorescence Microscopy for the Analysis of DNA Double-Strand Breaks

Published: July 31, 2023

Abstract

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

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 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…

Representative Results

Figure 1: Device for preparing the cytospins. Figure 2: Preparation of the cytospins. Two cytospins of each sample are prepared by centrifugation of 1.0 x 105 cells at 300 x g for 10 min. One cytospin is used for γH2AX immunofluorescence staining, and another cytospin is utilized for …

Açıklamalar

The authors have nothing to disclose.

Materials

Diagnostic microscope slides Thermo Scientific ER-203B-CE24 Microscope slides
Megafuge 1.0 R Heraeus 75003060 Tabletop centrifuge
Cytospin device
Lid Heraeus 76003422 Lid for working without micro-tubes
Cyto container Heraeus 75003416 Cyto container with 2 conical bores
Clip carrier Heraeus 75003414 Carrier for holding a cyto container and a slide
Support insert Heraeus 75003417 Support insert for holding a clip carrier
Paraformaldehyde Sigma-Aldrich P6148 Fixation agent
Phosphate-buffered saline Sigma-Aldrich D8537 Balanced salt solution
Chemiblocker Merck Millipore 2170 Blocking agent
Mouse monoclonal anti-γH2AX antibody (JBW301) Merck Millipore 05-636 Primary antibody for detection of γH2AX
Polyclonal rabbit anti-53BP1 antibody (NB100-304) Novus Biologicals NB100-304 Primary antibody for detection of 53BP1
Alexa Fluor 488-conjugated goat anti-mouse antibody Invitrogen A-11001 Secondary antibody
Alexa Fluor 555-conjugated donkey anti-rabbit antibody Invitrogen A-31572  Secondary antibody
Vectashield mounting medium Vector Laboratories H-1200 Contains DAPI for staining of DNA
Axio Scope.A1 Zeiss 490035 Fluorescence microscope
Cool Cube 1 CCD camera Metasystems H-0310-010-MS Camera system for digital recording
Isis software Metasystems Not applicable Microscope software

Etiketler

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Bu Makaleden Alıntı Yapın
Immunofluorescence Microscopy for the Analysis of DNA Double-Strand Breaks. J. Vis. Exp. (Pending Publication), e21465, doi: (2023).

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