COX-2 Staining Mystery

From:Greg Dobbin

Hello All,

I am having trouble with staining for COX-2 in dogs. I am getting 
widespread staining in many cell types especially epithelial even in 
normal healthy tissues). My sections are formalin fixed, paraffin 
embedded. I quench in 3% hydrogen peroxide (in distilled H20) for 
15 mins, followed by 20 mins., heat retrevial  (steam and citrate). I 
then block with normal serum (actually, DAKO Universal Blocking 
Solution), for 20-30 mins. The primary antibody (rabbit polyclonal to 
murine COX-2) is applied and incubated overnight at 4 C at a 
dilution of 1:400. I then use the DAKO Biotin Blocking System, and 
a then the DAKO LSAB-2 Steptavidin kit, (I don't  
work for DAKO, honest!). DAB is my chromagen.

My deletion controls (ie no primary Ab) are clean. It seems as 
though the rabbit anti- mouse COX-2 has something in it that is 
reacting with the dog tissues.  I also stained some human tissues 
and got similar results, but perhaps not as severe.

I found a "Brief Communication" in Vet Pathology (38:116-119; 
2001) that discusses expression of COX-2 in canine renal 
carcinomas. The authors used a rabbit anti-human COX-2 (which I 
may end up buying yet!), however don't mention antigen retreival 
per se, but speak of permeabilizing the tissues using triton and 

I am sorry for the length of this message. Let me sum up:

a) What is causing the (presumably) false positive staining?
b) is permeabilizing a form of antigen retreival or does it serve 
another purpose?
c) the contact person for the article I mentioned is Kristina M. 
Stanfield, Searle/Pharmacia, Skokie, Illinois. Does anyone out 
there know Kristina well enough to offer me her e-mail address?

Thanks for your patience. Have a great weekend!
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

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