Indirect immunofluorescence makes it possible to detect DNA repair proteins, photos of spatial and temporal recruitment and it helps interrogate Proteinprotein interactions at the site of DNA damage. Following DNA damage, DNA repair proteins are recruited to the DNA insult while their concentration increases locally, and they form groups called foci that can be visualized by indirect immunofluorescence on fixed samples. This technique can be used to detect protein foci, as well as to quantify colocalization of the present in cells.
This can help explain the sequence of events required for complex formation and for DNA repair. particular proteinprotein interactions can be entailed from such experiments. Demonstrating the procedure will be Barbara De La Pena, a very talented Postdoc Photo from my laboratory Begin by growing 40, 000 HeLa cells in each well over 12 well plate with an 18 millimeter round glass coverslip to 80%confluency.
After exposing the cells to four gray gamma irradiation, wash them twice with one milliliter of PBS. Then remove the PBS completely and add 200 microliters of NDB to each well. Incubate the cells for two minutes at room temperature then remove the NDB.
Keep in mind that incubation time can vary depending on the cell line, but generally should not exceed two minutes. Wash the cells with one 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 four degrees Celsius. Then remove the PFA and add one 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 two hours at room temperature or 16 to 18 hours at four degrees Celsius.
dilute the primary antibody and dilution buffer and vortex it until well mixed in a humidity box and here 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 two hours at room temperature.
After the incubation wash the cover slips three times in PBS for one minute per wash. dilute the secondary antibody and dilution buffer and vortex it until well mixed apply 10 microliters of secondary antibody to each coverslip as previously described and incubate it for two hours at room temperature protected from light. Wash the cover slips three times with PBS and once with water for one minute per wash.
Then mount them onto glass slides with a glycerol based mounting media containing DAPI seal cover slips with transparent nail polish and let them dry for 20 minutes. Place a drop of immersion oil onto the 60 x 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 type as roundtrip and 512 by 512 image size. In the PMT panel set mode to VBM average to frame and sequential scan to line.
Next set the dye and heat detectors set channel one to DAPI and SD one, channel two to alexafluor 488 and HSD three, and channel three to alexafluor at 647 and HSD four select on to 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 to 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. Open the analysis software then press the batch tool window and select the images to analyze. Go to the analysis tool window and select projection, which will display the maximum intensity projection from 15 slices.
Under the input output setting, select the created batch and output folder. Press process for images to be processed and export the images as TIFF files. Perform nuclear quantification with cellprofiler according to manuscript directions.
Cells not treated with the radiation exhibit very few gamma H2AX expo sign. In the absence of essential DNA repair proteins, gamma H2AX accumulation can be observed at DNA breaks. Accumulation of unrepaired breaks can lead cells to become pre hypotonic indicated by solid gamma H2AX nuclei.
Following irradiation, nuclei exhibit a large number of double stranded breaks to which gamma H2AX localize is extremely rapidly. While few if any foci are observed in the absence of a radiation the number of foci was quantified at one two four and 16 hours post a radiation and for the NOA radiation control. Depending on the biological question raised and the type of data required, different plotting options should be considered Colocalization can be investigated by multiplexing primary antibodies raised in different animal species and using secondary antibodies, are labeled with distinct fluorophores.
Superposition of green and red, gives rise to yellow hotspots, the two proteins of interest are present in the same pixels. Quantitative analysis of colocalization can be achieved by an object based approach or by a statistical approach that performs intensity correlation coefficient based analyses. A combination of primary antibodies raised in different animal species, can be used in this protocol.
Make sure that the antibodies are compatible and will not interfere with each other. You need to appropriately set the secondary antibody to be used against each of the primary antibodies and consider a particular spectral overlap when selecting the fewer force to be used. Colocalisation or proteins indicate possible direct interactions.
This can be verified by granular precipitation in cells, or by direct put down using purified proteins in vitro.
