[Histonet] RE: Nova Red/VIP substrate
When it comes to double staining, Vector Nova Red chromogen would not
be my choice for combining with DAB. Nova Red is not like AEC, it's
more brownish-orange and doesn't contrast that well with standard DAB.
You may try to shift the brown-yellow color of DAB reaction product to
more blueish, by adding nickel- or chromium-salts.
The combination of standard DAB with VIP gives a better contrast than
Both these chromogen kits are fully monkey-proof, don't worry about
If you perform this sequential double staining (with two HRP
activities involved), please keep in mind the following:
* it's not going to work for the observation of co-localization by
mixed-colors, just for two different cell populations (or
different cellular compartments) only.
* it's obligatory to apply DAB reaction product in the first
staining sequence! DAB reaction product is the only known
chromogen that effectively "cover" immunoreagents used in the
first staining sequence. This sheltering effect is the key
mechanism for preventing unwanted cross-reactions with reagents
used in the second staining sequence.
* sequential double staining works satisfactory if the first primary
(and secondary) antibody is well titrated. If the first
antibody (or secondary) is used too concentrated, the sheltering
effect from the DAB reaction product will not be effective.
I do hope I didn't scare you off performing double staining!
Chris van der Loos, PhD
Dept. of Pathology
Academical Medical Center M2-230
NL-1105 AZ Amsterdam
phone: +31 20 5665631
fax: +31 20 6960389
Date: Thu, 7 Jul 2005 15:45:59 +0200
From: louise renton
Subject: [Histonet] Nova Red/VIP substrate
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Could those of you in the community who have used these reagents from
Vector Labs for double labeled IHC reactions please comment on their
ease of use & contrast when used in conjuction with DAB substrate.
Bone Research Unit
University of the Witwatersrand
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