[Histonet] problem with "foggy" DAPI staining

From:Sarah Clatterbuck Soper

Hi all,

First I want to thank you all for being such a great resource.  I learn
so much from this list!  But now I have a question of my own.  I've been
having a problem for a while now, and I haven't been able to figure out
what is going on.  I hope you can supply some ideas.  :-)

I'm a graduate student working with mouse testes.  My usual procedure is
to dissect out the testes, fix overnight in Bouin's, process over the
next night (1 hour per station), embed, and section at 6 microns.  I
then PAS-H stain or perform fluorescence immunostaining.  If I am
immunostaining I always counterstain with DAPI.  This procedure works
perfectly for wild-type adult testes.

For juvenile mice or for our mutant mice, however, my DAPI staining
looks terrible.  It is fuzzy and diffuse looking, as if it is out of
focus.  It's like looking at the DAPI though a fog.  If I PAS-H stain
these sections they look fine.  Immunostaining still works.  It's just
the DNA staining that looks really crummy.

I've had wild-type and mutant testes processed side by side all the way
through; the wild-type looks great and the mutants look foggy.

Obviously, the difference must have to do with the size of the
tissues--the mutant testes weigh 25-35 mg, or about 1/4-1/3 of the
wild-types.  So I'm guessing I'm over-fixing or over-processing or
something.  I tend not to think it is over-fixing, as I've forgotten
some WT testes in Bouin's and processed them weeks later, and they still
look ok.  So my guess is over-processing, though is seems sort of
bizarre to me that processing would screw up the DNA somehow.  Does this
seem likely to you?  Do you have any other ideas?

Thanks so much for any help you can offer!


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