Re: ISH on murine embryos

From:Mikael Niku

I don't see why you should use cryosections for ISH.
Paraffin sections of paraformaldehyde-fixed samples work well -
and are probably safer for the RNA also. You just need to
optimize the pretreatments (proteinase, and/or microwave),
but this shouldn't be a significant problem since the
embryonic tissues are all so similar (as compared with
adult tissues, which may need differing pretreatments).

I'm routinely using PFA-fixed paraffin sections even
for genomic ISH, which I think is more demanding for
pretreatments, as the genomic DNA is tightly packed in
the chromatin. Microwave pretreatment is a must here,
combined with proteinase.

I have previously done mRNA ISH to PFA-fixed mouse embryos,
either whole-mount or paraffin sections, and proteinase
treatment alone seemed to be sufficient for them.

   Mikael Niku             URL:
   University of Helsinki  Dept. Basic Veterinary Sciences
       - Mitäkö mieltä olen länsimaisesta sivistyksestä?
         Minusta se olisi erinomainen ajatus!
                                              - Gandhi

----- Original Message -----
From: "Gayle Callis" 
Sent: Monday, January 14, 2002 6:51 PM
Subject: ISH on murine embryos

> >  Entering a new realm in my laboratory.
> >mRNA ISH is going to be done on mouse embryos, frozen sections.  Another
> technician will be doing the sectioning, and in my estimation, the
> cannot be kept RNAse free, too many use it, and I am not willing to clean
> up after people who tend to be a tidge sloppy.  They can either take their
> chances on doing this or
> >
> >1.  Fix the embryos, cryoprotect, then section - from what I have read in
> the past, many people already do this type of method.
> >
> >2.  Section fixed/cryoprotected embryos, use Cryojane Tape Transfer -
> which seems to be a way of controlling contamination AND keep the section
> on the slide during the stringent steps in the method.
> >
> >3.  Section unfixed embryos with Cryojane, to try and minimize
> contamination, section loss?  How successful or is anyone doing this
> 4.  Try and do paraffin sections on these tiny specimens?  They indicate
> this may not be as sensitive for ISH as frozen sections, maybe with their
> probe??? I am not sure why they think this, seen some good work out there
> on formalin and paraformaldehyde fixed tissues, certainly easier to handle
> for me.
> >
> >Are people out there using the Cryojane to do any ISH???  If so, are
> any modifications one needs to do with this.
> >
> >Trying to get moved into my new lab, the maxibroom closet, not looking
> forward to working in a tiny space nor walking across a street to get to
> toilets, dishwashing and distilled water supply. Time will tell on my
> of mind on this one.
> >
> >
> >Thanks
> >
> >Gayle
> Gayle Callis
> Histopathology Supervisor
> Veterinary Molecular Biology - Marsh Lab
> Montana State University - Bozeman
> 19th and Lincoln St
> Bozeman MT 59717-3610
> 406 994-6367
> 406 994-4303 (FAX)

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