This protocol provides a step-by-step procedure to detect radiation-induced foci of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam. Immunofluorescence imaging is a sensitive method and provides visual evidence for the appearance of repair pathway proteins in foci in response to DNA damaging-agents, like ionizing radiation. Neutron-mixed beam is used in boron neutron capture therapy, BNCT, and the radiation-induced DNA damage response has not been fully established.
Our protocol can also be useful for analysis of biological effects at the cellular level due to radiation by other high-LET beams, like protons using proton beam therapy or carbon ions using hadron therapy. After irradiation, remove the medium from the attached cells, and wash them once with 2.5 milliliters of PBS. Then fix the cells with one milliliter of 70%ethanol for 10 minutes.
To permeabilize the cells, remove the ethanol, and wash them with 2.5 milliliters of PBS. Gently add one milliliter of 2%Triton X-100 in PBS to cover the coverslips, and incubate the cells for five minutes at room temperature. Wash the cells three times with PBS, and block the permeabilization with one milliliter of 2%BSA diluted in PBS.
Then incubate the cells for a minimum of 30 minutes with the 2%BSA. To stain the cells, add the proper amount of primary antibody diluted in PBS with BSA, as recommended in the text manuscript. Then cover the Petri dish, and place it in a plastic box with moisturized lignin to maintain humidity.
Incubate it for 30 minutes at 37 degrees Celsius. After the incubation, perform three washes with PBS, and add the proper amount of secondary antibody. Return the dish to the plastic box with moisturized lignin, and incubate it for at least 30 minutes at 37 degrees Celsius.
Then repeat the washes with PBS, and add 100 microliters of DAPI diluted to a final concentration of one microgram per milliliter to counterstain the nuclei. Incubate the cells for a maximum of two minutes at room temperature, and repeat the washes with PBS. After the last wash, remove the PBS, and gently put a coverslip on top of the mounting medium, avoiding the formation of air bubbles.
Seal the edges of the coverslip with nail polish, and allow the mounting medium to harden before performing fluorescence microscopy. Gamma-H2AX foci, which are standard markers for DNA double-stranded breaks, were detected in neutron-mixed, beam-irradiated, and non-irradiated colon cancer cells. The foci appear as distinct fluorescent dots.
A higher diameter of gamma-H2AX foci was observed in the irradiated cells. Moreover, a single track of a high-LET alpha particle was detected crossing the nuclei. Immunofluorescence microscopy was used to detect representative repair proteins Rad52 and DNA-dependent protein kinase.
A high mean value of DNA-dependent protein kinase foci diameter was measured at DNA breaks after irradiation in comparison with control cells. Clustered DNA double-stranded breaks were observed in irradiated cells as more complex, larger, and clustered foci with higher intensity. The most important stage of immunofluorescence staining is fixation of cells and permeabilization of the cell membrane.
These steps are required to allow antibodies to easily penetrate both into those cells and intracellular repair proteins. This procedure gives information about the activation of each repair pathway at a cellular level. The results should then be confirmed at a molecular level using an assay, such as real-time PCR.
This technique makes it possible for researchers to explore radiation-induced DNA damage response, which has not been fully determined in the case of different beams using anticancer therapies.
