Histonetters,

I guess I'm confused, and would like Vector Labs to clarify.  My understanding of 
peroxidase is that the enzyme mediates a redox reaction that results in the 
formation of a covalent bond.  In the case of DAB, the substrate reacts with 
itself to form a long-chain polymer that is insoluble.  In the case of the 
tyramides, the substrate forms a covalent bond with nearby amino acid residues 
(tyrosines, if I remember correctly).  So why would the Nova Red substrate result 
in a product that is water-soluble?  Is the covalent bond that is formed labile in 
some milieus like glycerol?  I understand that this may be true of some FITC 
adducts.  Or are there two components in the kit that undergo an HRP-mediated 
reaction resulting in a charged product as suggested by John Kiernan?

I'm thinking about buying this product, but more information on the nature of the 
reaction product would be extremely helpful so that I know how to tweak it to make 
it work for my particular application (double-labeling of in situs in whole mount 
embryos).

Sometimes companys shoot themselves in the foot by keeping things too, too secret.

Karen Larison in Oregon      


Date:          Wed, 30 Jun 1999 16:08:33 -0600
From:          Gayle Callis <uvsgc@msu.oscs.montana.edu>
Subject:       NovaRed discussion results
To:            histonet@Pathology.swmed.edu

Histonetters, 

Turns out Vector's NovaRed is NOT compatible with aqueous mounting media.  You 
must dehydrate, clear and use a permanent xylene, toluene or Vectors
Vectamount media.  What I saw before fading occured (my booboo!) was 
impressive for my mousie CD markers on frozen sections.

Two reponses indicated washing with distilled or pure water was done, with
Vector response 45 min in running tap still maintained chromogen color. 

It should also be noted the specification sheet indicates Vector
Vectamount is not compatible and should not be used with Clearite 3 clearing.
Use Richard Allans mounting media (toluene based) instead or use xylene to 
clear with Vectamount.  Kathy Rohrbach indicated she had used the no no
combination of Clearite 3 then Vectamount.  Hopefully, she will not have
any long term problems and let us know if she does.  Interesting how we all
try something a bit different!

As for proprietary formulations, I can live with this situation, I am
interested in results that are superior even with a "secret" reagent. 
As far as legalities on such a thing, lawyers can strike up a wheely dealy
with vendor to find out very privately or with a reasonable explanation
to satisfy the courts, and let the vendor have their "proprietary day". OR 
medical legal gurus in-house can give legal guidelines on avoidance
of these issues.       

In this day and age, proprietary reagent usage is so widespread, it would
be hard to NOT find kits without this kind of reagent for IHC use and 
ultimately used for staining on some tissue involved in a court case 
on down the road.  If one doesn't like proprietary products, or absolutely
has to know what is there, then the choice is clear.  Don't use them, been
there, done that.  If it is a make it yourself issue, will have to use another.
Am finding many commercial products (proprietary or not) pretty darn
cost effective these days less time consuming with cleaner, superior results.

I am having one of those days, folks!  Yak, Yak!

Gayle Callis