Re: in situ
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From: | Kevin R Gray <grayk@bms.com> |
To: | "Hawkins, Hal" <hhawkins@SBI.UTMB.EDU> |
Reply-To: | |
Date: | Tue, 20 Jul 1999 07:45:37 -0400 |
Content-Type: | text/plain; charset=us-ascii |
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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