the cells were treated with 25uM of camptothecin for 3h then washed and fixed with 4% formaldehyde for 10min. Permeabilization was done with 90% methanol for 5 min @ RT. Blocking was done for an hour with PBS containing 0.5% BSA, 0.5% tritonX100. primary antibody incubation was done for 2h @RT. secondary antibody incubation was done for 1h @RT. I've tried two different primary antibodies #4895 from CST and #611366 from BD biosciences. Does anyone have experience working with these antibodies for immunofluorescence? Please share your experience.
Hi Sanjeev,
Have you thought about the following possibility? Did you confirm with western blot that there are good levels of Mre11 in the cell line you are working with? If the Mre11 levels are low in that cell line, it is quite possible that cells mediate the DSB repair without activation of MRN complex, therefore without formation of Mre11 foci.
Please take a look at this paper to support this idea and also you might find conditions to standardise the staining protocol to visualise Mre11 foci in the cells.
http://www.jbc.org/content/278/22/20303.long
Hope it helps!
Satish
hi Satish Bodakuntla
Thanks for the response. The western blot shows good levels of Mre11, in fact there is induction after DNA damage. But antibodies are not working in immunoflurescence experiments under my conditions I guess.
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