[Histonet] BrdU/DAPI and BrdU/hematoxylin counterstaining

[Ana Florentin]

Hello everybody!

I am trying to use the BrdU staining to look at proliferation in frozen rat aortic sections.

Detection of incorporated BrdU, by using monoclonal antibodies directed against BrdU, is only feasible in single stranded DNA. Therefore this technique requires a pretreatment to denature double stranded DNA. I am using 2 M HCl at 37ºC for 15 minutes and I obtained positive labeled cells in thymus (used as positive controls) as long as in the experimental aorta sections.

My interest is to count de BrdU positive cells and to quantify them as mean percentage of positive cells. In this regard, I need to easily discriminate the labeled cells from the unlabeled nuclei in the same microscopic fields. 

My problem is that I can not perform a double immunofluorescence staining BrdU/DAPI of the nuclei. The HCl pretreatment made the DAPI staining giving only a fuzzy blue background, thus I was unable to detect the nuclei.

I also tried the immunoperoxidase staining (which also worked for BrdU) and counterstaining with hematoxylin, with the same pretreatment, results and…problem. I know from literature that the hematoxylin counterstaining should work, even if there is the HCl pretreatment. But, in my hands, the supposed hematoxylin stained nuclei could not be clearly identified.

I would be really grateful for any suggestions or comments.

Thank you and all my best!

Anna 



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