I am doing in situ TUNEL staining on mouse tissue sections. I see a fair number of bright TUNEL-positive, DAPI-negative events in many sections.
I have seen in some papers that cells lose DAPI in late-stage death but still stain for TUNEL.
I just want to verify whether I should count these TUNEL-positive, DAPI-negative events as real.
The TUNEL assay is supposed to transfer a fluorescein-12-dUTP at 3´-OH DNA ends. In my opinion if you do not see DAPI you probably have a false positive signal.
I think you would still see DAPI positive cells in TUNEL stained cells. You may need to increase your DAPI staining concentrations or exposure time on the blue channel. You might need to have negative control where you have TUNEL unstained (without adding TUNEL reagents), but DAPI-stained cells. This would help you to see what you are seeing is a true signal with a red channel/TUNEL stain (or whatever fluorescent signal TUNEL is given with your kit). Hope this helps.
Thanks to you both! After doing some reading, I realized that these assays are notorious for false positives so that's probably the issue. I actually did run a negative control and saw nothing with those sections. So, I think the TUNEL itself is nonspecifically staining the slide all over.
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