Immunofluorescence Microscopy for the Analysis of DNA Double-Strand Breaks

To visualize specific DNA damage response proteins at DNA double-strand break, DSB, sites using immunofluorescence microscopy, begin with microscope slides containing human mononuclear cell monolayers with DSBs. Fix the cells with paraformaldehyde.

Treat with a non-ionic detergent, permeabilizing membranes for accessing intracellular targets. Add a protein-containing blocking solution, blocking cells' non-specific binding sites. Incubate with primary antibodies that bind to the target response proteins.

Treat with a secondary antibody mixture tagged with different fluorophores to bind specifically to their respective primary antibodies attached to the proteins. Stain DNA with DAPI. Add mounting medium; mount the coverslip.

During fluorescence microscopy, the incident light beam reaches the excitation filter allowing light of a specific excitation wavelength to pass through. Upon reaching the dichroic mirror — a beam splitter — the excitation light selectively reflects towards the objective lens, which focuses the light onto the sample.

The incident excitation light excites the fluorophore's electrons to a higher energy state. On returning to the ground state, the electrons emit lig