Encyclopedia of Experiments: Cancer Research
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Transcript
- Ionizing radiation induces different types of DNA damage, such as single-stranded breaks or SSBs and double-stranded breaks or DSBs. Single strand breaks or SSBs occur when the radiation damages the bases and cleaves the DNA backbone, while double strand breaks, DSBs, develop when two SSBs occur on opposite DNA strands. The first step of DSB formation is the phosphorylation of the H2AX histone, which can be visualized by immunofluorescence staining.
To perform immunofluorescence staining, add primary antibodies to a sample fixed on a cover slip placed in a Petri dish. The antibodies bind to phosphorylated histone protein gamma-H2AX. Incubate the sample and wash it with PBS to remove unbound antibodies. Next, add secondary antibodies carrying fluorophores, which recognize primary antibodies and bind to them. Add the desired amount of DAPI fluorescent stain to counter-stain the nuclei.
Gently mount the cover slip on a glass slide with a mounting medium and observe cells under a fluorescent microscope. The gamma-H2AX foci appear as distinct fluorescent dots in the irradiated cancer cells. In the following protocol, we will perform immunofluorescence staining of irradiated human colon cancer cell lines to identify radiation-induced DNA repair foci.
- After irradiation, remove the medium from the attached cells and wash them once with 2.5 milliliters of PBS. Then, fix the cells with 1 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 1 milliliter of 0.2% Triton X-100 in PBS to cover the cover slips and incubate the cells for five minutes at room temperature. Wash the cells three times with PBS and block the permeabilization with 1 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 1 microgram per milliliter to counterstain the nuclei.
Incubate the cells for a maximum of 2 minutes at room temperature and repeat the washes with PBS. After the last wash, remove the PBS and gently put a cover slip on top of the mounting medium, avoiding the formation of air bubbles. Seal the edges of the cover slip with nail polish and allow the mounting medium to harden before performing fluorescence microscopy.
