Re: insitu hybridization techniques for frozen sections

From:Marcus Andrews

Hi Terry- I've been doing in situ's off and on for the majority of my 
science career, using both oligo probes and riboprobes.  I've never 
used proteinase K in any of my protocols for frozen sections- during 
the fixing process, there is nomrally a delipidation step which 
normally facilitates probe entry into the cells prior to the 

Our lab is quite interested in looking at how different stressors 
affect specific mRNA levels, therefore ISH is a reasonable choice as it 
allows you to look more carefully at particular tissue regions, rather 
than accept an overall mRNA level such as you would get from a Northern.

I realize this has been quite a general reply- I hope it's helped! ;)


On Thursday, January 9, 2003, at 06:12  PM, Johnson, Teri wrote:

> Me again...
> I'd like to pick your collective brains again.  This time I'm looking 
> for
> information regarding doing ISH (RNA probes) on frozen section.
> Does anybody out there do them routinely, and if so, why?  Does one 
> still
> need to use Proteinase K to make the nucleic acids more accessible to 
> the
> probe?  What is your fixative of choice?  What other modifications to
> "standard" ISH protocols are made for frozen sections?  Anything else
> I've left out?
> Thanks for your advice!
> Teri Johnson
> Managing Director Histology Facility
> Stowers Institute for Medical Research
> 1000 E. 50th St.
> Kansas City, Missouri  64110
   Marcus Andrews, PhD.
   Department of Physiology,
   Faculty of Medicine,
   University of Toronto,
   1 King's College Circle,
   Toronto, Ontario,
   M5S 1A8


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