Patrick-

Remember to wear gloves or finger cots when handling bocks, sections,
slides or anything else of importance to mRNA insitu hybridization.  Human
finger tips are LOADED with RNAses.


Margaret







Kevin R Gray <grayk@bms.com> on 07/19/99 07:09:06 AM

To:   "Patrick M. Haley" <pmhales@cybergap.net>
cc:   Histonet <HistoNet@Pathology.swmed.edu> (bcc: Margaret
      Gondo/GeneMedicine)
Subject:  Re: in-situ question




Patrick,  based on my experience, when sectioning for ISH, the area
surrounding
the microtome or cryostat should be an RNAse-Free zone.  I use only
autoclaved
instruments and spray all equipment and storage containers with RNAse Zap
(Ambion).   As for blades for cutting I have found that using new blades
directly from the package is sufficient enough.  I use to process the
blades
through an RNAse Zap wash followed by 100% EtOH and allow to air dry but
following good results this way then tried blades straight from the box and
got
the same results.  Always wear new gloves and definitely clean the cutting
surface between blocks (70% EtOH w/DepC treated water) so as to not
transfer RNA
from a different tissue/organ/species.   Also, for paraffin slides, make
sure
the water bath is used with DepC treated water.  To get around this,  I
found
that rinsing bath first with RNAse Zap and then using MiliQ water gives
good
results as well.
I also prefer to use DAKO ISH slides (silanized).  These seem to work best
for
optimal results as well as keeping sections on slides.  Of course, other
slides
do work well but make sure they have never been touched by human hands (use
a
new box).
One final note,  do not make shortcuts when just starting out.  It may take
longer to do it the right way, but ISH takes time.  Remember what takes
longer,
doing something right once or repeated it two or three times?
Kevin Gray
Bristol-Myers Squibb
New Jersey
"Patrick M. Haley" wrote:
> I am posting this for a friend running in-situ via a kit method.
> They are wondering how critical the RNAse precautions are during
sectioning?
> Taking an educated guess, I told her very critical, (unless you want to
> destroy what you are trying to demonstrate). This person may be trying to
> take some short-cuts.
> Any thoughts or experiences about this subject?
>
> Many thanks!!
>
> Pat Haley
> HTN, inc.
> www.histology.net