RE: mouse alpha-smooth-muscle-actin immunohistochem

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From:"Matthew Ogdie" <mogdie@innogenex.com>
To:<histonet@pathology.swmed.edu>
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Content-Type:text/plain; charset="iso-8859-1"

Hello Tamara,
'Mouse-on-mouse' IHC can be performed effectively and with very little
difficulty.
The background, ofcourse, generally results from the secondary antibody
reacting with endogenous mouse Ig.
Typically, there are two approaches to blocking the cross-reactivity between
anti-mouse secondary antibodies and endogenous mouse immunoglobulins.
1) Block the endogenous immunoglobulins with an Fc blocker.
2) Pre-label the primary antibody.
Option 1) occasionally works, but it is not a consistent method [ monovalent
Fab fragments(Fc blockers) do not immobilize very well and, consequently, do
not always block the Fc region of Ig in the mouse tissue].
Option 2) works extremely well.
I would recommend either optimizing a pre-labeling procedure for your mouse
monoclonals or simply purchasing one of the 'Mouse-on-Mouse' IHC kits that
are available on the market.
It is probably more cost effective, and definitely much easier, to buy one
of the commercial kits. Just make sure you choose one that employs a
pre-labeling procedure, as opposed to an Fc blocker.
Needless to say, I would recommend the InnoGenex iso-IHC kits. I have
enclosed a manual from one of the iso-IHC kits(the protocol is on Page 3).
The procedure should clarify my comments.
Best Regards,
Matthew Ogdie
InnoGenex
(925)543-1414
http://www.innogenex.com



-----Original Message-----
From: Tamara Pereira [mailto:tamaraP@qimr.edu.au]
Sent: Tuesday, May 02, 2000 7:00 AM
To: 'HistoNet Server'
Subject: RE:mouse alpha-smooth-muscle-actin immunohistochem


Dear histonetters

Does anybody know of a supplier of alpha smooth-muscle-actin (aSMA)
antibody in any animal other than mice?

In our lab we are trying to look at aSMA expression in mice by
immunohistochem but we get a lot of background staining since our primary
(anti-aSMA) antibody is raised in mice. The antibody works fine with other
tissue (human, rat).  We are now searching for antibodies raised in other
animals but have had no success so far.  Meanwhile we are trying to
optimise our protocol to reduce the background (extra blocking etc), does
anyone have any tips on how best to do this?



Tamara  Pereira,
Hepatic Fibrosis Group
Clinical Sciences Unit
Queensland Institute of Medical Research
Brisbane, AUSTRALIA
Ph: 7-3362-0173
Fax: 7-3362-0191
email: tamaraP@qimr.edu.au




-----Original Message-----
From: Tamara Pereira [mailto:tamaraP@qimr.edu.au]
Sent: Tuesday, May 02, 2000 7:00 AM
To: 'HistoNet Server'
Subject: RE:mouse alpha-smooth-muscle-actin immunohistochem


Dear histonetters

Does anybody know of a supplier of alpha smooth-muscle-actin (aSMA)
antibody in any animal other than mice?

In our lab we are trying to look at aSMA expression in mice by
immunohistochem but we get a lot of background staining since our primary
(anti-aSMA) antibody is raised in mice. The antibody works fine with other
tissue (human, rat).  We are now searching for antibodies raised in other
animals but have had no success so far.  Meanwhile we are trying to
optimise our protocol to reduce the background (extra blocking etc), does
anyone have any tips on how best to do this?



Tamara  Pereira,
Hepatic Fibrosis Group
Clinical Sciences Unit
Queensland Institute of Medical Research
Brisbane, AUSTRALIA
Ph: 7-3362-0173
Fax: 7-3362-0191
email: tamaraP@qimr.edu.au







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