Re: in-situ question

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From:Kevin R Gray <>
To:Histonet <>
Date:Mon, 26 Jul 1999 12:57:54 -0400
Content-Type:text/plain; charset=iso-8859-1

Dorit,  I must respond to your message.  Are you recommending not wearing gloves for RNA
in situ hybridization(ISH)?  First and foremost,  gloves should always be worn in the lab for
safety issues.  For RNA ISH, even if you treat all solutions with your product (protect RNA),
the RNAses from your hands can effect any number of steps during the ISH run.  Negative
results can never have RNAse contamination ruled out as well if you do not wear gloves.  I
personally would never invest the time for ISH and place all my trust in one product.  Gloves
should be worn if for no other reason than for at least peace of mind.

Kevin Gray


> Instead of treating water with DepC, autoclave slides or even work with gloves, etc., just add "protect RNA" to your solutions. This is a new product from Sigma (No. R7397) that has been tested for in-situ, and is much cheaper than RnaseZap.
> Dorit
> ----------
> From:  Margaret Gondo
> Sent:  ιεν ωπι 19 ιεμι 1999 15:46
> To:  Kevin R Gray
> Cc:  Patrick M. Haley; Histonet
> Subject:  Re: in-situ question
> 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 <> on 07/19/99 07:09:06 AM
> To:   "Patrick M. Haley" <>
> cc:   Histonet <> (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.
> >

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