Immunofluorescence Microscopy for the Analysis of DNA Double-Strand Breaks

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.