[Histonet] inconsistent DAPI staining

[Joanne Whitehead]

This board is a great resource - I hope this problem has also been
encountered by some knowledgeable people here!

Sometimes I see huge variation in the DAPI signal when I view my
tissue sections under the confocal microscope, and I'm wondering if
this can be an artifact of poor freezing, as the sections that look
the worst tend to be the ones I had trouble sectioning and show the
worst morphology. 

At first I thought it was a problem with my mounting medium: I ordered
a hard-set vectashield with DAPI, and while I was waiting for the
order I borrowed a bottle of Prolong Gold from Molecular Probes. The
Prolong Gold worked fine, I just had to seal the coverslips with nail
polish to prevent damage to the sections. When the vectashield
hard-set arrived my first sections showed really inconsistent DAPI
staining - some regions nice & bright, others really weak. Generally
it the the outside edge of the section that is well stained, and the
interior faint. I thought perhaps the polymerizing of the mounting
medium happened too quickly, so that it would harden around the
tissue, which was still a bit wet from the last wash, and not
distribute evenly. So next time I air dried the slides really well
before mounting, but then had problems with air bubbles getting
trapped in the mounting medium, and still not good staining into the
middle of the sections. 

I talked to the Vectashield rep, who didn't have any good suggestions,
but did replace the bottle with a normal non-polymerizing DAPI medium.
But the problem of seeing DAPI staining only at the edges of the
sections is getting worse, and have since had the same problem with
the Prolong Gold too!

The DAPI I use only as a counterstain to make the pictures look nice,
but I wonder if the DAPI isn't evenly distributed, then perhaps the
flourescence protectant is also not evenly distributed, which would
undermine my quantitative analysis of the Cy3 signal I'm looking at.
Yesterday's slides were the worst of all - not only was there almost
no DAPI signal (expect a very tiny margin around each section), but
the Cy3 signal was almost invisible too, hence the concern about the
fluoroprotectant. I usually wait 2 days before analysing my slides,
as the antibody seems to continue to redistribute itself, giving
lower backgound over a day or 2, so I need to know that the
fluorescent signal is protected. I used to put the slides immediately
at -20C after mounting, but with the hard set I had to leave them at
4C so it could polymerize properly.

Help! Has anyone else dealt with this problem? What is the general
wisdom regarding drying (or not) the slides before mounting? Does it
have any effect? The consistency of my staining has been steadily
decreasing with the morphology of my sections - I hope I can solve
both problems soon!

Thanks very much.
Joanne
Institut Curie, Paris