Hello everyone,
I am performing immunocytochemistry (ICC) with 53BP1 antibody on treated HAP1 cells in order to identify 53BP1 foci within the cell nuclei. Recently, I have figured out that after staining the cells I get many foci around the nuclei (then supposed to be the cytoplasm) rather than inside them. Since I expect to find accumulation of 53BP1 only inside the nuclei in correspondence of dsDNA break spots, this situation seems uncommon for me.
I though that maybe it was due to improper blocking step, which I perform with 3% BSA in PBS for about 4h before adding primary antibody overnight at 4 °C.
However, I have followed the same procedure many other times on the same cell type always for 53BP1 foci staining, but I never had such problem.
If anyone has any technical suggestion or idea about this situation it would be very appreciated.
Thank you in advance.
53BP1 foci in nucleus mean that it detected double strand break. Ongoing DNA damage resposne means translocation of 53BP1 from cytoplasm to the nucleus. Therefore when you see the protein in cytoplsam, there is no DNA damage?
I do not think that this is the case, otherwise I should see cytoplasm foci in the untreated samples which show none or very few nuclear 53BP1 foci. These samples are treated with a molecule which induces dsDNA breaks, and the number of cytoplasmatic foci increase together with an increasement in the concentration of such molecule used for the treatment.
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