I'm doing BrdU staining in a cell line. I've treated the cells with 1.5 ug/ml of BrdU for 1h. Then I fixed them with 2% PFA for 15min, denaturated with HCl 4N for 10min RT, washes in PBS to neutralize. Permeabilization was done with 0.2% Triton X for 5 min and blocking 1h with 1% of BSA in PBS. Incubation with 1ary antibody against BrdU for 1h at RT ( BD Pharmigen produced in mouse), washes in PBS 0.05% Tween 20 and incubation with 2ary Alexa 568 anti-Mouse IgG (H+L), washes and mount with Vectashield. I only can see like dots in the cytoplasm of the cells but no clear nuclear staining. Has anyone experienced the same problem? Do you think that it may be a problem of BrdU incorporation or the inmunofluorescence protocol? Thank you very much in advance.
Hi Dear Yaiza, i have worked with BrdU several time. i think there is no need to use Antigen retrieval in Cell line. try one more time without retrieval. Fixation in 4%PFA for 20 min, wash 3 times 5 min , Permeabilization with 20% Tween PBS for 20 min, wash3*5 min, Blocking with Goat or other appropriate serum (20% Serum 0.2% Triton PBS+ BSA) for 20 min, extract Blocking solution, and don't wash in this step, then add primary antibody which has been prepared by 5% serum PBS for over night, after that, wash 3*5 min, and add secondary antibody in 5% serum PBS for 1 h. at the end, wash and watch under microscope .
Hi Yaiza,
I would recommend to include a sample untreated with BrdU to rule out antibody artifacts. The antibody may be binding nonspecifically. Permeablization may be incomplete as well. Remember that you not only have to permeablize the plasma membrane, but the nuclear envelope as well. I also recommend spinning down your secondary antibody prior to diluting, as aggregates of the conjugated fluorophore can often lead to increased background in staining.
Hi Yaiza,
I think your step with HCL 4N is unnecessary. Try the same protocol without it and you should avoid the issues you had.
Dear Majid and Benjamin, thank you very much for your suggestions, I will try without HCl treatment. Dear Jessica, thank you very much also. The secondary is already spun down. I can increase the time of permeabilization.
Hi Yaiza,
I am wondering if you finally found a solution to your problem ? I am facing the same when trying to do a BrdU stain on tissue sections.
Thanks
Dear Benoît, I changed many things but I couldn't fix the problem. To circunvent it, I have passed to Edu and FACS sorting. I don't know if this may be work for you, but if you manage to find out how to solve the problem with BrdU, please, let me know, I'm curious about it.
Hello Colleagues
Wondering if this thread of conversation is still active. I could use some help in discerning false +ves in BrdU signal in brain sections prepared from animals exposed to BrdU in-utero. thank you
Dear Jemal,
to circumvent my problem I changed BrdU to EdU incorporation followed by FACS detection as I did not find why BrdU was not working good enough and because by FACS it was easier to count automatically the cells. My cells were in culture so it was easy to treat them with EdU. I have never tried these methods in sections but I think you can use the advice from Majid, Jessica and Benjamin or maybe someone else has experience with sections... Good luck!
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