Greg,
you might want to try putting some dog normal serum in with your blocking
serum, i use normal serum from the animal being stained at a
concentration of 10%, block with this before the primary antibody for 20
min. and then again before the secondary for 5 min., this might help with
your non-specific binding problems.  don't use more that 10% dog serum or
you might block everything.
patsy

Greg Dobbin wrote:

> 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
> saponin.
>
> 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
> >
> >
> >
> >
> >
> >
> 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