In brief, I have been labeling my cells with 50uM BrdU over >2X the doubling time of the cells. Thereafter, I would wash the cells 4X with 1XPBS before fixing them with 4%PFA. The cells are then permeabilised with 0.5% Triton-X, 3% BSA in 1XPBS. The DNA are hydrolysed with 2M HCl at room temp for 45 minutes followed by 37degC for 15 minutes. The acid is then neutralised with 0.1M sodium borate buffer pH 8.5 for 30 minutes at room temp. I then follow up with my normal immunofluorescence staining protocol, where I block with 3% BSA in 1X PBS pH7.4 before subjecting to staining with my primary antibody, secondary antibody, cell mask and DAPI.
However, I do not get any strong BrdU signals in the nuclei of my stained cells.
Does anyone have recommendations on how I can improve my protocol for it to work?
To those people who have succeeded at doing BrdU staining for confocal microscopy, is there any anti-BrdU antibody to recommend? This is because I am also suspecting the quality of my antibody.
Hi Joanne,
my impression is that you are being a bit too harsh with the HCl treatment... On cells it should be quite quick the DNA hydrolysis. I would limit to 30 min at RT or 10 min at 37 C... You could also use a 1 or 1.5 M HCl solution instead of 2 M.
On cells you can also try a different approach. First fix and permeabilize the cells, then hydrolyze the DNA with 0.07 M NaOH for 30 min at RT and neutralize as above.
Concerning the labeling protocol itself, normally you should pulse the BrdU incorporation for max 30 min and then change the medium and grow the cells for one or two cell cycles in standard medium. If you change medium to the cells to add the BrdU, make sure you have prepared a prewarmed aliquot containing the BrdU and don't wash cells with PBS or you'll slow down the cell growth.
Good luck.
Thank you Dr Mohamed Mahdy, I will check out the protocol you provided and see how I can use it.
Dr Pietro Pilo Boyl thank you very much for your input. I will consider your advice and test out the use of NaOH as well as a less harsh HCl treatment. May I clarify what you mean by pulsing the BrdU incorporation? Do you mean to say that the cells should only be treated with BrdU containing media for 30 minutes, after which the media should be changed to non-BrdU containing media?
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