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
>