[Histonet] Cox-IHC in uterus

[Greg Dobbin]

Hi Jocelyne,

Here is my 2 cents worth:

a) For the Ab's that are not working, try incubating the primary 
overnight in the fridge before giving up on them.

b) For the one with excessive background, try adding (up to) 1% 
bovine serum albumin (or normal bovine serum) to the primary 
(make sure you treat your negative controls the same). This might 
adsorb out the (or some of the) non-specific rx.

c) If you really want to try the procedure without the universal kit, go 
for it. Just use goat anti-mouse or rabbit anti-mouse conjugated 
with peroxidase diluted to approx. 1:200. I do think however you will 
be sacrificing some sensitivity in the test.

Good luck.
Greg

Date sent:      	Sat, 15 Jan 2005 05:24:16 +0100
From:           	Leclerc Jocelyne <leclercj <@t> vetanat.unizh.ch>
To:             	<histonet <@t> lists.utsouthwestern.edu>
Subject:        	[Histonet] Cox-IHC in uterus

> Good morning!
> I have problems with performing COX-2-immunohistochemistry on bovine uterus.
> 
> I haven`t found an adequate antibody yet. Those that I tried showed
> excessive unspecific bindings (Merck Frost) respectively didn`t bind at all
> (BD Transduction labs, mouse monoclonal clone 33).
> Additionally,  employing a Vectastain ABC Universal  Elite Kit (rabbit and
> mouse IgG) I have severe problems with background, especially in the
> perimetrium and connective tissue.
> Seems like the secondary antibody cross-reacts with the bovine tissue
> although I did peroxidase block (0.3% in methanol for 30 min), avidin-biotin
> block (Vector), the normal serum which came along with the Vectastain kit
> and sometimes tried an additional protein block (Dako).
> 
> I would be very happy if anyone of you out there could share the experience
> with me and help with finding an antibody an eliminating the
> "universal-kit-problems".
> Thank you very much.
> 
> Jocelyne Leclerc
> 
> 
> ____________________________________
> Jocelyne Leclerc
> Institute for Veterinary Anatomy
> Vetsuisse Faculty, University of Zurich
> Switzerland
> 
> 
> 
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~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Greg Dobbin
Pathology Lab
Atlantic Veterinary College, U.P.E.I.
550 University Ave.
Charlottetown, P.E.I.
Canada,  C1A 4P3
Phone: (902)566-0744
Fax: (902)566-0851
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Making a living is getting; making a life is giving.