Encyclopedia of Experiments: Biological Techniques
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Transcript
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 light at a longer wavelength, collected by the objective lens, and passed back to the dichroic mirror.
The mirror allows the emitted fluorescence light to pass through the emission filter, which allows only the desired fluorescence light to reach the detector. Process the images. Analyze the different colored fluorescent foci to visualize the co-localization of the DNA damage response proteins at the DSB site.
