Immunofluorescence Microscopy for the Analysis of DNA Double-Strand Breaks

Prepare two cytospins with mononuclear cells of the patient samples by centrifugation of 1 x 10⁵ cells for each preparation. Fix the cells with 200 microliters of 4% PFA for 10 minutes.

After fixation, wash the cells gently three times with 30 milliliters of PBS for five minutes each on a shaker. Then, permeabilize the cells with 200 microliters of 0.1% Octoxinol-9 for 10 minutes. Wash the cells gently three times with 30 milliliters of 5% blocking solution for five minutes each on a lab shaker. Block the cells in 30 milliliters of fresh 5% blocking solution for one hour.

The fixation and permeabilization of the cells with 4% paraformaldehyde and 0.1% Octoxinol-9 preserved the gamma H2AX and 53BP1 foci distinctly.

Incubate one preparation of the cells with a mouse monoclonal anti-gamma H2AX antibody and the other preparation of the cells with a mouse monoclonal anti-gamma H2AX antibody and a polyclonal rabbit anti-53BP1 antibody overnight at 4 degrees Celsius.

The use of proven anti-gamma H2AX and anti-53BP1 antibodies is highly recommended.

After incubation, wash the cells gently three times with 30 milliliters of 2% blocking solution for each five minutes on a lab shaker.

Next, incubate the first preparation of the cells with an Alexa488-conjugated goat anti-mouse secondary antibody diluted 1 in 500 in 2% blocking solution. Incubate the second preparation of the cells with an Alexa488-conjugated goat anti-mouse secondary antibody and an Alexa555-conjugated donkey anti-rabbit secondary antibody diluted 1 in 500 in 2% blocking solution.

Wash the cells gently three times with 30 milliliters of PBS on a laboratory shaker. Remove the PBS and mount the cells with mounting medium. Cautiously, put a coverslip on top of the mounting medium, so that no air bubbles are embedded. Wait at least three hours for the mounting medium to harden before analyzing the cells by fluorescence microscopy.

Finally, analyze the gamma H2AX and 53BP1 foci in the cell nuclei with a fluorescence microscope equipped with filters for DAPI, Alexa488, and Cy3 during imaging at a 100x objective magnification.