RE: In-situ hybridization (ISH) on paraffin sections

From:jeff and wanda gray <jefwan@InfoAve.Net>

I have successfully run mRNA ISH on sections from our "hold" slide boxes,
and have used controls that have been sitting in the control box for several
weeks.  I find that using RNAse precautions in the dehydrating and
pre-hybridizing steps to be sufficient.  I use the BioGenex kit to run my
ISH, and use their RNAse preparation included in the kit.  I'm getting
excellent EBV stains with an RNA probe.  Feel free to contact me with any
Wanda Shotsberger Gray HT (ASCP)
Charleston, SC

 -----Original Message-----
From: 	Miriam Schroeder []
Sent:	Tuesday, September 19, 2000 3:41 PM
Subject:	In-situ hybridization (ISH) on paraffin sections

Hi.  I was wondering if anyone has experience with
in-situ detection of mRNA in paraffin-embedded tissue.
 In particular, I wondering about the necessity of
RNase-free methods in the tissue PREPARTION
(processing, cutting, etc.)  I have several protocols
for the actualy ISH itself, so I know that once the
slides have been deparaffinized, all the solutions
must be RNase free.  However, none of my protocols
talk about the earlier stages (the tissue

It would seem to me that I would not need to take
special care to prepare "RNase-free" fixative because
the RNase enzyme itself (if present) would ALSO be
fixed, which would serve to eliminate its activity.
(????)  Similarly, I would think if there were any
RNases present in my alcohols, xylenes, and paraffins
that in these organic conditions the RNase would
assume a very different conformation than under normal
physiologic conditions and thus it also would in
effect be inactivated.  (And that the solidified
paraffin would physically immobilize the RNase enzyme
- ???).

If I must use RNase free methodology during these
early steps, what do I do during the cutting process?
Must I clean my microtome with some sort of RNase zap
product & use new brushes & RNase free tools?  Do I
need to use RNase-free water in my water bath for
flotation of the sections?  What about the "Chrome
Alum" adhesive I use in my water bath?  Do I need to
be concerned whether it is RNase free?

If anyone has experience with these aspects of tissue
preparation for ISH, I'd greatly appreciate your
advice.  In particular, I would be interested in
hearing from people who have done ISH in a
reasearch-type setting, with a poorly characterized
probe.  (vs. a clinical / diagnostic type situation
where things would be better standardized & these
"unknowns" already answered).  (For example - if there
is LOTS of mRNA present, maybe a tiny bit of RNase
activity wouldn't be such a disaster.  (???)  But if
the "message" is not so "strong" it might be a greater

I would also be curious to hear from people working in
clinical (hospital) labs about any experience you
might have with ISH.  Is ISH commonly done for
diagnostic purposes?  If so, how do you prepare the
tissue in the hospital-type setting?  I am guessing
that the tissue would not be initally processed &
handled in any special way (just processed with all
the other routine tissues), and only later (after H&Es
have already been done) would a decision be made to do
ISH.  Maybe this type of work would even be sent out
to a reference lab?  I am just curious because my
pathologist worked in a clinical setting before coming
to industry so we try to emulate the stringency of
clinical procedures.  (So if in the clinical situation
you don't take special precautions, then my boss would
probably think we didn't need to, either.)

Finally, all of my histology texts are rather old,
they definitely do not include chapters on in-situ
hybridization.  Can anyone recommend a good reference
for in-situ which is geared for histologists (esp.
concerning the tissue processing questions I have).
In addition to any reference texts you might know of,
if you also know of on-line resources, I'd appreciate
hearing about them.  (I'll be doing this tissue
preparation next week so I doubt I'll be able to get
ahold of a new histology text before then.)

Thanks for all your help!
Miriam Schroeder
Research Associate
Berlex Biosciences
Richmond, CA

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