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Hello,

This is with regard to the use of nuclear dyes other than DAPI. I have
been using Sytox Green from Molecular Probes to look at the nuclei of
Drosophila tissue. The company says that Triton precipiates the dyes and
phosphate buffers should not be used. So I use PIPES buffer with EGTA
and magnesium for all my dilutions and washings.

But still I find that the dye is not stable. The Sytox Green fades
very quickly sometimes as soon as 1 day after making the slides (even
before I have had a chance to scan them!). Also there is a lot of
background staining.

Has anyone faced the same problems with this dye? Is there any other
buffer or protocol that I could use to make the dye last longer?

Thanks in advance,
Bela

*******************************************************************************
Bela S. Desai
Graduate Student
B-228, Department of Biological Sciences,
Tata Institute of Fundamental Research,
Bombay, India
400 005

On Tue, 22 Apr 2003, Goran Ocklind wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Try SYTO 13 from Molecular Probes. I have used it for nuclear staining of
> Caco-2 cells on polycarbonate filters with a very good result.
>
> Göran Ocklind
>
> -----Ursprungligt meddelande-----
> Från: Confocal Microscopy List [mailto:[log in to unmask]] För
> Daniel Scharlau
> Skickat: den 22 april 2003 09:54
> Till: [log in to unmask]
> Ämne: DAPI on filters!
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi everyone,
>
> this question is not really a confocal related question, but I thought
> someone out there might still be able to help. I have tried to stain the
> nuclei of  Caco-2 cells grown on polycarbonate filters using DAPI. The
> dye seems to bind to the filter or the filter has a high fluorescence in
> the UV range. At least I get a very weak specific signal and a very
> brigth background. I have used the same protocol with coverglass grown
> cells and got perfect results. Does anyone experienced something similar
> and can suggest solutions to this problem (special protocol, other
> dyes...)? I would be very grateful for any kind of help.
>
> Daniel Scharlau
>
> --
> Daniel Scharlau
> Max-Planck Institute for Molecular Physiology
> Dep. Epithelial Cell Physiolgy
>
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> 44227 Dortmund
> Germany
>
> Phone: +49 (231) 133-2204
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