Hello all,
	I am experimenting on goat myocardium for characterizing the
conduction velocity during ischemic and nonischemic conditions. for this I
need to stain the myocardium, immediately when it is removed from the goat
and to detect the cell viability. I am using the Trypan blue and TRiphenyl
tetrazolium chloride stains  for this purpose. Does anybody know the
procedure.

                       Suvarna Yogesh Udgire,
                  Indian Institute of Technology Bombay
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On Sat, 15 Jun 2002, rueggp wrote:

> 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
>
>
>
>