Why is DAPI counterstaining used AFTER all other antibodies are attached to cells during immunohistochemistry protocols?
I ask because for my particular protocol, it would be easier for me to do the DAPI staining right after I do the fixation step, but what I've seen online is that most people do the DAPI step after everything else. Is there a reason this is done and can the DAPI stain be done before other antibodies are tagged to my cells?
(I'm using HS936 cells or "melanoma" from ATCC.org and I'm staining with CD45 and MART-1 using conjugated quantum dots in various other colors otherwise)
I can think of simple reason. DAPI staining takes only minutes and is a fluorescent compound. If your other procedures takes 6 hours, why you want to expose your DAPI six hours by passing in different solutions and light. Also I am not sure while passing through many solutions, it might or might fade.
Dapi is a nuclear stain as we all know. And it is used as a counter stain. After we localize a particular antigen on cell we counterstain with dapi to make sure that only the cell has taken the stain and not an artifect or other thing. So in the end we take a merge photo. Also as DAPI is a florescent stain we do it at the end so that the florecence may not be lost.
One practical consideration: the antibody staining solutions and washes between them often contain detergents (e.g. tween or triton x100), while staining with DAPI is done in the absence of detergents and is usually preceded by a couple of washes with PBS only. I suppose the reason for using DAPI last could be to minimise the number of washes.
Ah, so hypothetically it could be done as long as there isn't too much fading? The reason I ask is that I'm using quantum dots and exciting everything simultaneously using a DAPI excitation source and filter on our microscope. The reason I'm doing this is that merging photos is time consuming since we are looking for one cell out of an entire 96 well plate potentially, so I need to be able to visually see the fluorescence simultaneously without having to merge the photos and look computationally.
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