Most immunofluorescence protocols I've seen apply DAPI as the last step in staining, following the application of primary and secondary antibodies.
If DAPI was added first, could it interfere with other antibodies' binding?
Hello Austin Morrissey
Most cells are colorless and transparent. So, no matter what your specific antibody is, whether it be fluorescent or not, it can be difficult to identify cell type and location. Therefore, counterstaining is important.
If you counterstain your sections post-immuno, it can help you to pinpoint the exact position of your positive cells. A counterstain also provides contrast so that the antibody-stained cells stand out more. Moreover, DAPI being a fluorescent stain, it should be added at the end so that the fluorescence is not lost during the washing steps.
Best.
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