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Graham,
I don't quite get it: Why do you expect Dapi-staining to be homogeneous
in the first place? It pretty much never is, except you totally destroy
the nuclear structure. Dapi stains AT but not GC, so AT-rich
heterochromatin is particularly bright but GC rich heterochromatin (e.g.
in horses and donkeys, Fig. 5 in doi 10.1007/s00412-008-0200-6,
http://www.springerlink.com/content/1342m1n1n4358g85/fulltext.html)
stays unstained.
The pattern you get very much depends on the species and also the cell
type you are looking at.
Generally, DAPI-dark areas are either low in DNA content in general or
in AT-bases in particular. If you want to find out which of the two, you
can costain with Propidium Iodide (after RNA-digestion) since PI is
intercalating and does not discriminate between AT and GC (to the the
best of my knowledge) but it also stains RNA, therefore the digest.
Steffen
On 04.09.2012 11:35, Graham Wright wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> *****
>
> Hello,
>
> We have DAPI stained nuclei in cultured mammalian cells and they are
> typically not homogeneously fluorescent, with obvious areas of very low
> intensity, such as the nucleolus, but also a patchy-ness in the rest of the
> nucleus with varying intensity throughout. For us this is most easily seen
> in confocal or deconvolved-wf slices. Does anyone know, or have any
> theories on, what these 'darker' areas are a result of?
>
> Some thoughts we had are regions relatively deficient in DNA, or
> heterochomatin rich regions? Or is it just a result of differential binding
> as a result of A-T rich regions?
>
> Any thoughts on this will be much appreciated.
>
> Thanks,
> Graham
>
> ---
> *Dr Graham Wright*
> Microscopy Unit Manager
>
> Institute of Medical Biology
> 8A Biomedical Grove, #06-06 Immunos, Singapore 138648
>
> E:
[hidden email]
> W:
http://www.imb.a-star.edu.sg/imu/>
--
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Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy
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