RE: ISH on murine embryos

From:Ralph Marcucio

Hi Gayle-

My lab routinely (3-4 times/week) performs ISH (35S-riboprobes) on 4%
para-fixed, paraffin-embedded avian embryos.  I have not encountered a
sensitivity issue with any of the probes I use.  I would imagine that unless
the mRNA species of interest is in very low abundance that paraffin would be
just fine.  Certainly, non-radioactive in situ hybiridzation is much less
sensitive unelss you employ enhancement techniques like tyramide.  I have no
experience with this since the radioactive in situs work beatifully.

We keep EVERYTHING RNAse-free, including our microtome.  If people are too
lazy to keep it to the required standard, they are not allowed to use
it...period.  Having said that, RNAse contamination does not seem to be a
problem.  THere are a couple of steps that are very sensitive to RNAse
contamination.  For example, after protease treatment, the mRNA is
particularly susceptible.  Nonetheless, we are VERY careful to avoid RNAse
contamination.  If you would like any further info. please let me know.

Ralph Marcucio

Ralph Marcucio, PhD
Department of Orthopaedic Surgery
University of California at San Francisco
San Francisco, Ca 94143

Phone: 415-502-4945

-----Original Message-----
From: Gayle Callis []
Sent: Monday, January 14, 2002 8:52 AM
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 cryostat
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 method?

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 there
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 state
of mind on this one.
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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