I have been trying to count the H2AX foci on my immunofluoresence experiments, and usually the whole nucleus is stained. How do I count those.. Or if due do excess damage the whole nucleus is stained, how do I add them to my data as a measure..Thanks
there should be a striking intensity in the nuclei with much damage (H2AX foci) as compared to that of background stain. you may want to reduce the intensity of the channel when viewing under microscope to identify the nuclei with true damage and count that. after counting make a graph of fractions that have formed H2AX foci.
hi azeez, i use software to count the gamma H2AX foci. for this olympus fluoview microscope helps a lot. to count the foci please follow the steps. set an ROI in one of the foci inside cell(nucleus). This intensity and higher intensity is selected and the weaker intensity as compared to ROI is deleted. Now you can clearly visualize the foci as the background stain is lost or negated. All the best.
hi Mohsina, the software is really helpful, since you do not have to adjust intensity and it can automattically compare intensity that are high above a threshold. thats really cool
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