Hi, I'm Ian Ma from the Epigenomic Medicine Lab head by Dr.Tom Caris at the Baker Eye, DI Heart and Diabetes Institute. I'm currently undertaking my PhD, which has a strong focus on the effects of radiation on Gamma H two A X, which is the molecular marker of double stranded breaks. Today I'll be demonstrating an experiment on the quantitation of Gamma H two A expo in response to gamma radiation in human cretes.
This immunostaining protocol can be used to monitor both initial DNA damage as well as DNA repair. This method is also useful for evaluating the efficacy of potential radiation modifying compounds. For instance, radio sensitizers, which are expected to increase DNA damage, will result in higher numbers of gamma H two A X four side, while radio protectors would have the opposite effect with lower numbers of ForSight detected due to a reduction in DNA double stranded braids.
Now let's start the experiment.Experiment. The cells used in this experiment are human cytes, which are prepared in chamber slides with 10, 000 cells seated in each well. Slides are incubated at 37 degrees in 5%carbon dioxide for three days prior to radiation.
Slice the transporter 2D irradiator on ice, then irradiated at two gray while controlled slides are left un irradiated. After irradiation slides are returned to the incubator for one hour, this incubation period allows us to look at initial DNA damage. Next comes the staining.
Media is first to build the wells carefully before the addition of 300 microliters of calcium chloride and magnesium chloride free PBS into it chamber. To rinse the cells slice the left on the plate shaker for five minutes before the PBS is gently tipped out. Next cells are fixed in a hundred microliters of 4%paraldehyde and left at room temperature for 10 minutes before being placed in a coton jar filled with PBS on the plate shaker for five minutes.
This wash step is repeated to give a total of three five minute washes. Following the wash, the slides are gently dried on absorbent paper. To remove excess PBS, the cells are then premiumized in a hundred microliters of 0.1%Try the next 100 for 10 minutes at room temperature After 10 minutes, the slides are washed in PB S3 times for five minutes each as shown previously.
Next, a hundred microliters of 1%BSA was added into which well and left for 20 minutes to block non-specific binding. After the blocking step, the BSA is shaken off and a hundred microliters of primary antibody, which is made up of a one in 500 dilution of anti phospho. Gamma H two X antibody is added into each well.
The slides are left at room temperature for one hour, followed by three five minute washes in PBS. Excess PBS is shaken off and a hundred microliters of secondary antibody, which consists of a one in 500 dilution of Alexa floor 4 88 gold anti mouse IgG is added into which well and left to incubate in the dark for 45 minutes at room temperature at the end of the incubation slides are washed in PB S3 times for five minutes each covered in foil to prevent photo bleaching. A hundred microliters of the nuclear stain.
Top row three is added into which well for 10 minutes, followed by two five minute washes. With PBS, the chambers are now ready to be taken off the slide. Gently take the chambers off by pulling upward and release the slide from its holder.
Tap away any excess moisture and allow the slides to dry before adding a drop of prolonged gold Antifa reagent. Slowly lower a cover slip onto the slide, taking care to avoid trapping air bubbles. Wipe away the access reagent and leave the slice in the dark at room temperature for half an hour.
Finally, apply nail polish along the sides of the cover slip to secure it and leave the slides at four degrees in the dark overnight. A confocal microscope is used to visualize the gamma H two A expo site formed. Settings are adjusted to obtain a sharp image with minimal background noise.
Images are required in the Z Series pattern with a step size of 0.5 microns to minimize loss of foci present in different planes of the nucleus. During analysis, individual planes are de convoluted and stacked to produce a maximum projected image to minimize the overlap of foci. To quantitate the number of foci generated, the meta of program is used, each nucleus is highlighted using the tools available on the program and each region of interest may be transferred onto the image with the gamma H two A X foci.
The program quantitate a number of foci in each cell after the threshold has been applied to exclude all background, and the information is locked into an Excel spreadsheet for further analysis. And that concludes our immuno setting protocol to visualize the initial D damage following exposure to carbon radiation. This sensitive assay can also be applied to other aspects of radiation induced damage involving double stranded brakes with high reproducibility and reliability.
Thanks for watching.I.
