I'm trying a whole mount assay with BrdU. I've read that people have issues with DAPI staining after treating their slides with HCl for antigen retrieval. This is a whole mount assay, as I've already said, so I'm wondering if it would affect my sample as well. Has anyone had any problems?
Hi Laura,
DNA denaturation implicates double helix separation, DAPI staining works by intercalation so the "strenght" of denaturation will surely impact the following DAPI staining.
You could surely try to optimize denaturation temperature and time to get a balanced result of DAPI staining and antigen retrival, or you can try a different retrival protocol for proteins. Citrate buffer and high temperature works fine for most proteins and don't affect strongly DAPI staining.
Best
I have done many BrdU assays and never saw anything strange with the appearance of the DAPI. The type of antigen retrival mentioned in the earlier comment (citrate++high temperature) is actually not useful for BrdU visualization. A typical protocol for BrdU retrival is 30 minutes HCl 2N (37 degrees C) + 20 minutes of re-equilibration with Borate buffer pH8 (and extensive washes afterwards). This might affect the appearance of nuclear DNA but should not prevent DAPI staining. Rather, fluorescent proteins (ex. GFP, RFP) and other antigens in your tissue might be affected and not detectable by primary antibodies. In case BrdU assay turns out not being compatible with your stainings, you may want to try the "new" generation proliferative assays such as EdU or IdU, which do not require HCl pre-treatment nor antibody-based detection.
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