Hi,
My question is this:  did you try it with only one round EDTA HIER?  My
first thoughts are that you did not, and that the problem is basically that
the tissue is being over-done for the 2nd round.  I have never done 2 rounds
of HIER for double labeling...... but I've never done double-labeling with
EDTA, either..... only citrate... but the one round works for me.
Bonnie Whitaker
UT--Houston

----- Original Message -----
From: Sebree Linda A. <la.sebree@hosp.wisc.edu>
To: 'C & J Haley' <haley@primary.net>
Cc: 'Histonet' <histonet@pathology.swmed.edu>
Sent: Wednesday, March 01, 2000 8:10 AM
Subject: RE: Double IHC staining question


> Hi Jane,
>
> I'm curious as to why you're doing 2 HIER procedures.  We've been able to
> get by with using the longer HIER procedure required and toning down on
the
> other antibody if the HIER procedure results in too intense staining or
> background staining.  I would recommend trying to do this or finding a
HIER
> procedure that is optimum for both antibodies.  I don't know if this will
> crisp up your second antibody or not but it might be worth a shot.
>
> Linda A. Sebree, HT
> University of Wisconsin Hospital & Clinics
> Immunohistochemistry/In Situ Hybridization Laboratory
> D4/218-2472
> 600 Highland Avenue
> Madison, WI  53792-2472
>
>
> (608)265-6596
> FAX: (608)263-1568
>
> > -----Original Message-----
> > From: C & J Haley [SMTP:haley@primary.net]
> > Sent: Tuesday, February 29, 2000 10:51 PM
> > To: HistoNet Server
> > Subject: Double IHC staining question
> >
> > Hello Everyone:
> >
> > I'm here to dip into the histo-wisdom pool again.  I've been doing some
> > double IHC staining.  This round of double staining involves two
> > antibodies requiring EDTA HIER.
> > Has anyone had experience with two EDTA requiring antibodies?  I've been
> > able to get staining, but the second antibody staining is not as
> > "crisp"  as I would like.  Any tips?  For those interested:  Yes, I am
> > actually doing two rounds of EDTA on the slides.  No, I'm not having any
> > section adhesion problems.  Yes, I've tried both alk. phos. and
> > peroxidase systems for the second round with the same results.
> >
> > Thanks in advance,
> >
> > Jane Haley
> > Barnes-Jewish Hospital
> > St. Louis, MO
> >
>