Chromogens / Vector Red + Correction

From:Miriam Schroeder <>

Sorry to be slow.  My reply to two messages:
(Also, a correction to my original post is at the end)

>Curious about the high background you said you
>experienced with the 
>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 
>Catherine "Katie" Bresee Bennett
>Sr. Research Technologist
>Lovelace Respiratory Research Institute
>Albuquerque, New Mexico

Katie:  No, I haven't tried an avidin/biotin block,
this had never occured to me.  Thanks for the
suggestion.  The strange background I've experienced
has never made sense to me.  My
Streptavidin/Peroxidase & Streptavidin/Alk Phos are
both from Zymed.  Since I DON'T have problems when I
do the Vector Red first, but I DO when DAB comes
first, do you think this indicates the problem is with
my Streptavidin/Peroxidase reagent?  (Maybe it doesn't
bind all the biotinylated secondary from the "first
round" of labeling?)  (By the way - at one point we
thought maybe it had to do with our secondaries -
whether I was using biotinylated Gt-anti-Rabbit or
biotinylated Gt-anti-Mouse.  But I've tried enough
different combinations to know that it has nothing to
do with this, the only consistent problem I've found
is in the order of the Vector Red vs. DAB.)

>I enjoyed reading your post.  I also love the
>reagent....or should I 
>say I
>"loved" the reagent.  Why?  Once a while, the color
>disappeared after 
>I know this happened to other people, too.  If you
>know the secret of 
>loosing the color, please inform me.  Thank you.
>Hiro Nitta
>BD Biosciences

Hiro:  I haven't experienced this problem yet (nor was
I aware of the potential for this problem), so if I
have  "secret" I don't know what it is.  Are you
specifically using the Vector brand reagent?  (Vector
Red)  I am not sure, but I suspect this reagent is
just Fast Red or something closely related.  At the CA
Soc Histology mtg. I learned that there are forms of
Fast Red which can be mounted in organic medium, and
other forms which cannot so must be mounted in aqueous
medium.  Maybe you're using the wrong mouting medium
for the brand of reagent you have?  (Vector Red CAN be
mounted with organic medium.)

NOW, for a correction to my original post:
>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
>have to proceed with next primary, biotinylated
>secondary, strep-peroxidase, DAB.)  ANYWAY, my point
>is that the reaction product "stayed put" even
>an antigen retrieval procedure.

After I read back over this, it didn't make sense to
me, I see I was off a bit with my details.  In fact,
my primaries WERE from 2 different species, but I
could not put them on together because 1 required
antigen retrieval & antigen retrieval was BAD for the
other.  Thus my overall procedure was:  1st primary,
biotinylated secondary, strep/alk phos, vector red,
antigen retrieval, 2nd primary, biotinylated
secondary, strep/peroxidase, DAB.  I incorrectly
stated above that after antigen retrieval I reacted
with the "second" seconary.

Miriam Schroeder
Research Associate
Berlex Biosciences
Richmond, CA

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