Re: Double IHC question with clarification

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From:C & J Haley <>
To:"Patterson, Noelle" <PattersonN@NMRIPO.NMRI.NNMC.NAVY.MIL>, HistoNet Server <>
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Amazingly enough, a good  DAB in the first round of IHC will NOT come out with
citrate or EDTA HIER.  Are you surprised?  I was. I've done several different
antibody combinations for other projects.  Some with citrate, some with EDTA and
some with nothing.  In this study,  I am doing the nuclear stain first.   I am
getting double staining, it's the second round of IHC for CD4 that's not as
pretty as I would like.  I'm just splitting hairs for photographic quality.
I've ordered a few different colored chromagens from Vector.  That's my next
round.  Vector Labs has a great web site with some very nice photos of multiple
staining and a general protocol.

Thanks to everyone else who has responded.  The Histonet is such a great

Jane Haley

"Patterson, Noelle" wrote:

> Just a thought.  Could it be that the second round of antigen retrieval is
> washing away the first substrate?  Have you checked that the chromagen from
> the first antibody is still there after the second round of HIER, and before
> the second antibody.
> Did you try different orders of the detection?  I would think that the
> nuclear antigen detection should be first, or you may be losing it.
> This is an interesting problem to me, and I will be very interested in the
> solution(s).  It is the research scientist in me.
> Noelle Patterson, M.S.
> Bethesda, Md.  20889
> stressed is desserts spelled backwards.
> > -----Original Message-----
> > From: C & J Haley []
> > Sent: Wednesday, March 01, 2000 9:00 PM
> > To:   HistoNet Server
> > Subject:      Double IHC question with clarification
> >
> > Hello again:
> >
> > Okay, so I didn't give enough information to get enough information.
> >
> > Linda, Patsy, Danielle:      I know, usually if they require the same
> > pretreatment, there's no need  for repeating it.  These guys need to have
> > their own round of EDTA HIER :( . Believe me, I've tried cutting one out,
> > altering one's time, etc.  I have optimized them separately.  Separately,
> > they look great.   But I need to have the double staining on the same
> > slide.    And that leads me to the next question.
> >
> > Cynthia:  So why would anybody need to put two antibodies that work best
> > alone on the same slide?  The answer is simply:  pesky Pathologist, pretty
> > pictures for a publication, and a nice little challenge.  I should  also
> > mention that one antibody is a nuclear marker and the other is a nice
> > little membranous marker.  Co-expression on the same cell  should make for
> > some nice photos. And to answer your  questions:  yes(substate),
> > yes(times/temps), and no because that shows that you know the many problem
> > parameters of IHC.
> >
> > Diagnostically, most double  immunostains are not practical.  For
> > researchers it can serve many purposes (co-expression, working with
> > material limitations, antibody specificity, etc.).  Also, most  Histotechs
> > (especially those on the Histonet)  like a challenge and a beautifully
> > stained slide. So, I'll continue to "tweak" my procedure and wait for more
> > responses or questions.
> >
> >
> > Thanks in advance,
> >
> > Jane Haley
> > Barnes-Jewish Hospital
> > St. Louis, Mo.
> >
> >
> >
> >

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