Hi Blerta,

You don't mention concentration and incubation time of camptothecin. It would be better if before doing your full high throughput experiment, you tried different concentrations and different incubation times of camptothecin to find the optimal conditions for induction of γH2AX foci.

Also, I think it would be a good thing to try one or two other DNA damaging agents (like bleomycin or hydroxyurea) that will induce γH2AX foci formation, so that you can exclude with certainty the possibility that there's something wrong with your primary or secondary antibodies.

Finally, maybe there's something wrong with the aliquot of camptothecin that you are using. Maybe try a fresh one.

Hope this helps

Cheers

Panos

Hi Kotsantis, first of all thank you for your suggestion. I did all preliminary experiments, as fixing the right nr of cells, type of drugs (bleomycin, neocarzinostatin, etoposide, and finally CPT in different concentrations, time of treatment that was from 24h to 30'). The problem could not be solutions that I used because they are all new. I am wondering if phosphatase enzymes reduce my signal by inhibiting H2AX phosphorilation. In immunofluorescence kit  it is not reported to add phosphatase inhibitor, we usually do it only in western blot.  

I am open for any kind of suggestion. Thank you again

May be you can do a quick western blot analysis for the expression of the same. Can you relate that signals to IF signals? You may want to check H2A level as well. I wonder whether you can use RAD51 or related marker from the same pathway.

Some times moisture level reduces during the cell fixation/ Ab incubation. This dries the cells significantly hence they do not retain sufficient Ab.

Lastly, you can add phosphatase inhibitor to the preparation, however you will be required to add extra controls in the experiment showing inhibitors are not tempering with reality of the signals.

yH2Ax are tricky ones.

Good luck