Catherine,
Just follow the directions included with the detection kit you are 
using. All you have to do is optimize the parameters surrounding 
the primary antibody, i.e. dilution, incubation temperature, 
incubation time and whether or not antigen retreival is necessary.

So start with the manufacturers suggested dilution and titrate 
around that (eg if 1:200 is suggested for IHC, try 1:50, 1:100,  
1:200, and 1:400). For incubation start with the simplest, 1 hr at 
room temp. If with these parameters you get no signal, try 
incubation overnight at 4 C (fridge). If still no signal, or siganal is too 
weak, try an antigen retreival method (assuming you are working 
with formalin fixed tissue??). For example, 20 mins. in a citrate 
buffer in a vegetable steamer. If after all this you still have no signal, 
I would try a different primary antibody, not all antibodies are 
created equal! I found that a polyclonal anti-COX-2 made to 
recognize human COX-2 worked well with my rat tissues whereas a 
polyclonal anti-COX-2 made to recognize canine COX-2 did not 
work.

Now if after Ag retreival you have too much signal (ie signal on 
deletion control) than the problem might be endogenous biotin 
(retreival enhances or accentuates endogenous biotin expression). 
Use a biotin block after retreival but before detection kit (I apply 
mine before the primary antibody). 

I hope this wasn't too elementary for you. If I over-simplified the 
explanation or if I was way off base on what you were looking for, it 
would be due to the lack of details in your e-mail request about 
what you are working with or what you may have already tried. 
Have a nice weekend and good luck.
Greg

Date sent:      	Fri, 12 Dec 2003 11:04:30 -0600
From:           	"Goeden, Catherine" 
Subject:        	[Histonet] COX-2 antibody
To:             	"'histonet@lists.utsouthwestern.edu'" 

> Hello everyone in Histo-land and Happy Friday!!
> 
> I am looking for a procedure using Biocare antibody for COX-2 and their
> detection kits.  I am in need of the step by step instructions and have been
> having difficulty finding the procedure.  Can anyone help?  Thanks in
> advance
> 
> _______________________________________________
> Histonet mailing list
> Histonet@lists.utsouthwestern.edu
> http://lists.utsouthwestern.edu/mailman/listinfo/histonet


~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
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.

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