Immunofluorescence Microscopy for the Analysis of DNA Double-Strand Breaks

Overview

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

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).

2. γH2AX and 53BP1 Immunofluorescence Staining

1. Fixation and permeabilization: Fix the cells with 200 µL of 4% PFA for 10 min. After fixation, wash the cells gently three times with 30 mL of PBS for 5 min each on a lab shaker. Then permeabilize the cells with 200 µL of 0.1% Octoxinol 9 for 10 min. Wash the cells gently three times with 30 mL of 5% blocking solution for 5 min each on a lab shaker. Block the cells in 30 mL of fresh 5% blocking solution for 1 h.
2. Incubation with antibodies
   1. Incubate one preparation of the cells with a mouse monoclonal anti-γH2AX antibody (1:500) and the other preparation of the cells with a mouse monoclonal anti-γH2AX antibody (1:500) and a polyclonal rabbit anti-53BP1 antibody (1:500) overnight at 4 °C.
   2. After incubation wash the cells gently 3 times with 30 mL of 2% blocking solution for each 5 min on a lab shaker.
   3. Remove the blocking solution and incubate the first preparation of the cells with an Alexa488-conjugated goat anti-mouse secondary antibody (1:500) and the second preparation of the cells with an Alexa488-conjugated goat anti-mouse secondary antibody (1:500) and an Alexa555-conjugated donkey anti-rabbit secondary antibody (1:500) for 1 h at room temperature.
3. Mounting medium: Put the slide with the cells in a bowl and wash the cells gently three times with 30 mL of PBS for each 5 min on a lab shaker. Remove the PBS and mount the cells with a mounting medium containing 4,6-diamidino-2-phenylindole. Cautiously put a coverslip on top of the mounting medium so that no air bubbles are embedded. Wait for at least 3 h for the hardening of the mounting medium before analyzing the cells by fluorescence microscopy.

3. Analysis of γH2AX and 53BP1 Foci

1. Fluorescence microscopy: Analyze the γH2AX and 53BP1 foci in the cell nuclei with a fluorescence microscope equipped with filters for DAPI, Alexa 488, and Cy3 during imaging at a 100X objective magnification. Record images with a camera and process the images with appropriate imaging software.

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 combined γH2AX and 53BP1 immunofluorescence staining.

Materials

Name	Company	Catalog Number	Comments
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