RE: [Histonet] Eosinophil marker

From:"UniPath IHC"

	I guess I should have made myself clear, and I admit I forgot to
sign my name before I sent off the email.  I'm an IHC tech working for a
private company.  However, I'm not just trying to get "free advice on the
internet".  I have been a member of the Histonet community for four years
and have enjoyed reading and contributing to this service.  I would never
use a response from Histonet as anything but advice or a hint in the right
direction.  As for who I work for, I have never been told that working for a
private lab as opposed to a hospital excluded me from Histonet. 
	I am working on a research project for a couple of doctors at the
National Jewish in Denver.  I am trying to doublestain eosinophils in skin
biopsies by IHC and am having no luck with the MBP antibody (Chemicon) on
routinely processed FFPE.  It works fine for me on frozen sections, and even
a little bit on tissue fixed in 4% PFA.
	I found a few emails in the archives regarding eosinophil markers,
but most people asking what I need to know had never been replied to, at
least not via the Histonet.  I have considered special stains to mark
eosinophils, but the doctors I am working with would like to use IHC.
	If I cannot make this antibody (MBP) work on FFPE then I need to
find another eosinophil marker that does.  Any help would be appreciated.

Thanks for your time,

Brianna Jackson, BS, QIHC
UniPath, LLC
Denver, CO

-----Original Message-----
[] On Behalf Of John Kiernan
Sent: Wednesday, October 27, 2004 9:28 AM
To: UniPath IHC
Subject: Re: [Histonet] Eosinophil marker

You identify yourself only as "Unipath IHC."
Why? Are you a commercial outfit asking for
free advice on the internet? If so, would
you sell services to clients on the strength
of someone's 15-minute response on a
listserver? Internet replies, including mine
(which follows) have no real value.  If you
do work for a client based on advice collected
from an internet enquiry you are asking to be

That said, I'll offer some advice. do not 
follow it without checking that it's based
on established and generally accepted 
knowledge. Compare 3 major textbooks. If
there are discrepancies, follow up the
references in your library. Get photocopies
of older papers by interlibrary loan if
necessary. That's one of the ways to get
details of techniques.

Antibodies are expensive. Eosin isn't, and it
stains the cytoplasmic granules of eosinophils 
strongly. If you stain with an alkaline (pH 9)
aqueous solution of eosin, the only red things
in animal tissues will be eosinophil granules,
Paneth cell granules (in the intestine) and
tails of spermatozoa (if present). I think this
is a complete list; someone, please correct me 
if I've missed something.

If you need a counterstain, use any
haemalum (Ehrlich, Gill, Harris, Mayer etc)
or a blue basic dye (eg toluidine blue at 
pH 4) according to your needs. Either of
these "counterstains" should probably be 
done before the alkaline eosin staining.

Dyes are cheaper and easier to use than
antibodies!  Immunohistochemistry is an
important family of methods, but it is 
expensive and is not needed for simple jobs
such as staining eosinophils. Your 
enquiry did include information about
the tissue you work with. Whatever it
is, you should not confuse eosinophils
with Paneth cells or sperm tails!
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
UniPath IHC wrote:
> Can anyone recommend an eosinophil antibody that works in FFPE tissue?
> _______________________________________________

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