RE: Chromogens

From:"Bennett, Catherine (Katie)" <>

Curious about the high background you said you experienced with the Vector
Red when used after DAB.  I use the DAB first then the Vector Red and
haven't seen this problem.  Have you tried an Avidin/Biotin block between

Catherine "Katie" Bresee Bennett
Sr. Research Technologist
Lovelace Respiratory Research Institute
Albuquerque, New Mexico

> -----Original Message-----
> From:	Miriam Schroeder []
> Sent:	Thursday, September 14, 2000 6:18 PM
> To:
> Subject:	Re: Chromogens
> Hi!  I would highly recommend Vector Red substrate, it
> is a substrate for the alkaline phosphatase enzyme. 
> (If you are currently using biotinylated secondaries
> the only change you would need to make in your
> procedures is to use streptavidin/alkaline phosphatase
> instead of streptavidin/peroxidase).
> Vector Red Substrate Kit, Catalog #SK-5100, $65;
> Vector Laboratories, Inc., Burlingame, CA;  Ph:650 697
> 3600;
> We regularly use this chromogen in double-label
> procedures in combination with DAB.  The only caveat I
> have is what I find to be an inexplicable phenomenon: 
> if the Vector Red labeling FOLLOWS the DAB labeling,
> there tends to be incredibly high background, the
> Vector Red seems somehow to even stick to the DAB so
> you end up with BOTH primaries being labeled red
> (=OOPS).  We've never had problems when we do the
> Vector Red reaction first, though.  (We really do love
> this chromogen, just make sure it PRECEDES DAB in your
> procedure.)
> The Vector Red reaction product seems very "hearty". 
> We have even done our first labeling reaction, then a
> microwave ANTIGEN RETRIEVAL procedure, and then
> proceded with the next biotinylated secondary /
> streptavidin-peroxidase & then DAB.  (OR, if both of
> your primaries were raise in the same species you will
> have to proceed with next primary, biotinylated
> secondary, strep-peroxidase, DAB.)  ANYWAY, my point
> is that the reaction product "stayed put" even through
> an antigen retrieval procedure.  Also, you can
> dehydrate & coverslip with an organic mounting medium
> like Cytoseal 60 or Permount.  This is a great
> advantage over something like AEC that requires an
> aqueous monting medium.
> Also, you cannot possibly confuse the Vector Red
> reaction product with endogenous melanin or
> hemosiderin pigments.
> Finally, the best part:  Vector Red is also
> fluorescent.  (Supposedly the organic mounting medium
> even IMPROVES the fluorescent qualities of the
> reaction product.)  This is a really useful feature
> because if you have absolutely any doubt about your
> staining, you can switch back & forth between
> brightfield & fluorescence.
> I hope this helps.  If you try the Vector Red, I'd be
> curious to hear how it works out for you!
> Miriam Schroeder
> Research Associate
> Berlex Biosciences
> Richmond, CA
> --- Patricia Karlisch <> wrote:
> > Dear Histonetters:
> >   We would like to do some double staining and are
> > looking for an alternate chromogen to stain with. We
> > use the standard DAB and Nova Red. The nova red that
> > we are currently using is giving a washed out
> > reddish color.  Do any of you know of a chromogen 
> > (and its supplier) that gives a vivid red color?  
> > Can you recommend anything else that will not
> > interfere with endogenous melanin or hemosiderin
> > pigments.  Thank you in advance.
> > 
> > Pat Karlisch, Team Leader
> > Geisinger Medical Center
> > Danville, PA  17822
> >
> > 
> > 
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