Re: insitu hybridization techniques for frozen sections


Here's a reference for a protocol that is very useful:  Whitfield, H.J. et
al., "Optimization of cRNA probe in situ hybridization methodology for
localization of glucocorticoid receptor mRNA in rat brain: a detailed
protocol", Cell Molec. Neurobiol. 10: 145-157, 1990.
No Proteinase K is involved;  there is some fixation during slide
preparation (4% formaldehyde/PBS for 5 minutes).  One important
consideration for ribo's is to DEPC-treat & then autoclave all supplies
(racks, buckets, etc.) & use DEPC-treated/autoclaved deionized water for all
solutions.  Best of luck!   Susan
----- Original Message -----
From: "Johnson, Teri" 
To: "Histonet (E-mail)" 
Sent: Thursday, January 09, 2003 6:12 PM
Subject: insitu hybridization techniques for frozen sections

> 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

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