Jane,

I'm still not getting this. Are you doing two HEIR methods consecutively and 
then doing the double stain, or are you doing HEIR, a stain and then another 
HEIR and another stain?

If it is the former you may simply be over treating the epitopes of the one 
that comes out fuzzy. If it is the latter it may be a steric hinderence 
problem (the first hindering the second) or an overtreatment problem.

It would probably be best to work towards one HEIR method that would 
retreive both, although that may take a lot more work.

Also, you if the compatabilities are suitable you could apply the primaries 
in a pooled solution and then the secondaries in a pooled solution rather 
than doing each separately and sequentially. that would reduce steric 
hinderance problems.

Tim Morken, B.A., EMT(MSA), HTL(ASCP)
Infectious Disease Pathology
Centers for Disease Control
MS-G32
1600 Clifton Rd.
Atlanta, GA 30333
USA

email: tim9@cdc.gov
       timcdc@hotmail.com

Phone: (404) 639-3964
FAX:  (404)639-3043


----Original Message Follows----
From: C & J Haley <haley@primary.net>
To: HistoNet Server <HistoNet@Pathology.swmed.edu>
Subject: Double IHC question with clarification
Date: Wed, 01 Mar 2000 18:00:03 -0800

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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