Until I create my strains with a GFP-tagged protein of interest, I've been doing immunofluorescence on yeast to look at this protein of interest examine its location.
After primary incubation, washes, secondary incubation, and more washes I subject the yeast to 10uL of DAPI at 1:1000 for 5 minutes at RT. Then, I wash 2 x 5 minutes with PBST (0.1% Triton-X 100).
Sometimes the DAPI staining produces a halo-like effect in addition to regular nuclear staining. There also seems to be a lot of cytoplasmic staining in some cells. I do not believe the nucleus was compromised. Does anyone know the cause of this?
Hello Kendra,
Do you mean as halo-effect that the blue stain is not located on a very limited space? It is due to that the DNA is not condensedn when the cells are not dividing, only when the DNA is packed into chromosomes the staining gets more localized. Make a transmission photo and one with the DAPI stain and make an overlay then you see if the stain is there where the nucleus is. Normally you should always make such a merged picture.
It also depends on the cell type, some cells have a very compact nucleus. The brigth spots in it is the nucleolus and the darker spot with lesser blue staining is the vacuole. I hope I have remembered correctly from histology ;-)
If a netlike or chessboard like structure appear on the cells then you have mycoplasma contanination due to the fact that DAPI is used to identify them based on the staining of their DNA when they are attached to the cell.
When the cell and nucleolus bounderies fit to your DAPI photo then it is a normal staining for your cells. Sometimes the pictures can a bit vary in which cell cycle the cells are you have in your focus.
I hope this helps you :-)
It is worth nothing that DAPI stains and accumulate in regions of DNA with condensed heterochromatin and highlight chromocentres.
The staining phenomenon is mainly due to internal reflection and scattering of emission rays from the DAPI stained yeast cell. The final border the emission rays has to penetrate is the plasma membrane and this also cause scattering of light, which will contribute to the halo phenomenon as you call it. The halo in the image is actually the boundary of the plasma membrane to its surrounding.
I have overseen that these are budding yeast cells and not human. Kenth-Arne´s answer is to right one.
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