Re: in situ

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From:Kevin R Gray <>
To:"Hawkins, Hal" <hhawkins@SBI.UTMB.EDU>
Date:Tue, 20 Jul 1999 07:45:37 -0400
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Hal,  I am not sure what you do for fixation and what your
hybridization/ post-hybridization temperatures are, but here are
my a few of my conditions and recommendations:

I fix cells in  4% cold pbs buffered paraformaldehyde for about
20-30 minutes
I do not use any proteinase k digestion.  My hybridization
temperature is 60 deg C.  My strongest and most stringent
post-hyb. wash is with 0.1X SSC/ 0.1M DTT @ 60 deg C.  For more
details please write me.  I would like to know your conditions to
better help.

Good luck,

Kevin Gray
Bristol-Myers Squibb
New Jersey

"Hawkins, Hal" wrote:

> We are starting a project in which we want to do in situ
> hybridization (for IL-8 mRNA) on cytospin preparations.
> So far, most of the cells are coming off.  Does anyone
> have a method that will allow us to keep the cells tightly
> adherent to the slide during the processes needed for
> hybridization?
> Thanks
> Hal Hawkins, UTMB Galveston

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