Re: Double IHC question with clarification
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From: | C & J Haley <haley@primary.net> |
To: | "Patterson, Noelle" <PattersonN@NMRIPO.NMRI.NNMC.NAVY.MIL>, HistoNet Server <HistoNet@Pathology.swmed.edu> |
Reply-To: | |
Content-Type: | text/plain; charset=us-ascii |
Noelle:
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
resource!
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.
> NN-TAB
> Bethesda, Md. 20889
>
> stressed is desserts spelled backwards.
>
> > -----Original Message-----
> > From: C & J Haley [SMTP:haley@primary.net]
> > 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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