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 …
Disclosures
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 |
Tags
Cite This Article
Immunofluorescence Microscopy for the Analysis of DNA Double-Strand Breaks. J. Vis. Exp. (Pending Publication), e21465, doi: (2023).