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1) http://products.invitrogen.com/ivgn/product/A10037?ICID==%3Dsearch-a10037 is my secondary antibody against mouse antibody. It is writing there is “a minimum cross reactivity” with rabbit antibody. So I think that there is no interference between two. It’s real that 53BP1-TRF1 co-staining is not the most good looking. But my lab used the combination of the same two secondary antibodies on other protein and apparently no cross reactivity was observed
2) I have tried PFA fixation for this co-staining. Unfortunately, the staining does not work. Because I need to examine a kinetic response on 10 days, I have to storage my samples before making staining. Maybe that PFA fixation is incompatible with 10days storage at -20°C… I don’t know.
3) Thank you so much. I will send to you an e-mail when I make the next staining.
Asked on Jul 19 2012
Thank you for your reply.
I hope the new vial will provide good results!
I am sorry that I have not seen the crossreactivity note on the datasheet of the secondary antibody.I might would nevertheless performa control slide using your settings/antibodies.
In regards to the PFA fixation, it might well depend on the cell and other antibodies, whether this is suitable. I do however not think that the storage is incompatible with PFA - you fixed the cells for about 10 minutes, washed them, and keep them in PBS/Azide in the fridge? This normally should work. In contrast to Methanol fixation, PFA fixation requires cell permeabilization after fixation. Maybe thepermeabilization step canbe improved,up to 0.2% Triton in PBS can be used for 10 minutes.
As said above, I hope the new vial will resolve the problem. Please keep us updated and do not hesitate to contact us also meanwhile, should you have any questions or concerns. I look forward hearing back from you!
Answered on Jul 19 2012