I fix cells with 4% PFA/sucrose for 15 min, and I block with 5% normal serum from secondary species. I recently started getting this issue, and I was able to successfully fix and stain one time without the issue.
There is some substance that is lying in between the areas with cells, which you can see from comparing the phase contrast to the fluorescent images. But, the Hoescht and green staining seems to indicate that there is some substance that is fluorescing on the cells.
Has anyone seen this happen before? I rinse with DI water before mounting the cover slips and it doesn't look like PBS crystals, so I don't think that's an issue.
I don't know why there's something between the spaces with cells that isn't fluorescing at all, but I have a few general suspicions:
1) Serum is sticking. I'm using really old serum (circa 2009) that has been aliquoted and stored at -20.
2) Cells are somehow drying out, although I aspirate and add solution in a sequential manner.
3) I use a vacuum to aspirate during washes and such, and possibly it's not strong enough to remove all the liquid.
4) I recently switched from VECTASHIELD non-hardening mounting medium to the hardening one.
In my esperience is the situation 2). some times I observed the same phenomenon when the cells were drying S......
Because your samples have been in a salty buffer through growth, fixing, staining, washing... why change it at the end? This seems like a miscibility problem. Keep the cells in the same buffer they've always been in my opinion. In fact, the best mounting medium I've ever used was just 90% glycerol in PBS.
Donald: The problem could be a combination of things, to include the suggestions above. In addition, this artifact - or one that looks extremely similar to it - we have observed when the mounting pressure on the coverslip is too high. Perhaps this, in combination with mounting media issues is a source of the artifact.
Vectashield Hardset outgasses, where as it cures, air pockets form under the coverslip. A big weakness (along with blue autofluorescence and quenching of far red dyes, like Alexa Fluor 647). Do you think what you are seeing may actually be gas pockets? If not, then I would suspect the old serum.
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