Immunofluorescence-Based Visualization of DNA Repair Protein Interactions
Trascrizione
Begin by growing 40,000 HeLa cells in each well of a 12-well plate with an 18-millimeter round glass coverslip to 80% confluency. After exposing the cells to 4 Gray gamma irradiation, wash them twice with 1 milliliter of PBS. Then, remove the PBS completely, and add 200 microliters of NEB to each well. Incubate the cells for 2 minutes at room temperature, then, remove the NEB.
Keep in mind that incubation time can vary depending on the cell line, but generally should not exceed two minutes. Wash the cells with 1 milliliter of PBS. Then, remove the PBS completely, and add 200 microliters of 4% PFA to each well for cell fixation.
Incubate the cells for 10 minutes at 4 degrees Celsius, then, remove the PFA and add 1 milliliter of PBS to each well. Remove PBS completely and then, add 200 microliters of blocking solution to each well, and incubate the cells for 2 hours at room temperature, or 16 to 18 hours at 4 degrees Celsius.
Dilute the primary antibody in dilution buffer, and vortex it until well-mixed. In a humidity box, adhere a piece of parafilm, and add 10 microliters of primary antibody in a single drop. Align one edge of the coverslip from the well with the drop, and slowly lower it onto the parafilm, spreading the liquid. Incubate the coverslip for 2 hours at room temperature.
After the incubation, wash the coverslips three times in PBS for 1 minute per wash. Dilute the secondary antibody in dilution buffer and vortex until well-mixed. Apply 10 microliters of secondary antibody to each coverslip as previously described, and incubate for 2 hours at room temperature, protected from light.
Wash the coverslips three times with PBS — and once with water — for 1 minute per wash. Then, mount them onto glass slides with a glycerol-based mounting media containing DAPI. Seal coverslips with transparent nail polish, and let them dry for 20 minutes. Place a drop of immersion oil onto the 60X objective lens, and use DAPI to locate the nuclei through the eyepiece.
For XYZ image acquisition, open the acquisition software and set the "Scanner type" as "Galvano", the "Scanner mode" as "Round Trip", and "512 by 512" image size. In the PMT panel, set mode a VBM, average a Frame, and sequential scan a Line. Next, set the dye and heat detectors. Set channel 1 to DAPI and SD1, channel two to Alexa Fluor 488 and HSD3, and channel 3 to Alexa Fluor 647 and HSD4. Select "ON" di "Z."
To adjust the live image, press the Live button on the Live Window and adjust the focus. Then, use the "PMT tool" window to set laser intensity, sensitivity, gain, and offset. For Z stacks, select Start a End and 15 Slices. Select the folder to save images, and press the LSM Start button to start acquiring. When finished, press the Series Done button to complete the image acquisition.
