I am imaging cells which have endogenous GFP signal and I stained them with DAPI in mounting medium. I use confocal ZEISS LSM800.
Some of the cell soma (looks like cytoplasm and processes) seems to have weak DAPI signal. Nuclei are nicely stained with visible chromatin pattern. Other cells without GFP signal have properly stained nuclei but there is no soma staining and also no hint of AF488 channel.
Is it possible that it is some weird type of channel bleeding or DAPI stained something in the processes?
DAPI could also stain RNA, so cytoplasm may have DAPI signal. You
can use RNase next time.
Dapi could stain microtubules if it is in excess. Likely you see only the stronger, bundled ones in processes.
Maria Gotkiewicz , DAPI has a very broad emission spectrum. It actually overlaps quite a bit with the GFP emission. It would be best to image sequentially. Note also that the laser used to excite DAPI can also excite GFP, so besides emission bleedthrough, there is also cross-excitation. I would also suggest that you don't use a mounting medium that has DAPI in it, instead use DAPI separately as a counterstain.
Johanna Marie Dela Cruz Hi, yes, it is done sequentially (first channel A, then B and then C). GFP is excited first, red afterwards ending with DAPI. The overlap is seen in one structure in the cell, and not in all of the pictures as well.
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