|From:||Miriam Schroeder <firstname.lastname@example.org>|
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
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
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
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!
--- Patricia Karlisch <PKARLISCH@geisinger.edu> 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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