COX-2 Staining Mystery
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
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!
Atlantic Veterinary College, U.P.E.I.
550 University Ave.
Canada, C1A 4P3
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