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From:Nicola Falconer <>
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Dear Paul

Yet again I'm voicing my opinion in favour of NovaRed, (and Vector in
general) and no I have no involvement with them other than as a satisfied
customer (although if they feel that I deserve a discount for supporting
them, then...). We have used NovaRed for some time and think it's great, in
fact, recently we run out (supply dept fault) and went back to Sigma Fast
Red, hope the NovaRed arrives soon.
The most common reasons for NovaRed dissolving seems to be either the buffer
is not the correct molarity and or pH, or you have acetone or similar
contaminating one of the solutions used after NovaRed has been applied

Hope this is helpful

John Auld
Royal Free Hospital

From: Paul Klosen <>
Subject: Antibody elution and NovaRed


I'd just like to share my last "misfortune" with NovaRed.
I'm currently running non-radioactive hybrizations combined with ICC. I
detected my non-radioactive ISH with TSA amplification and peroxidase
NovaRed as the final signal, and I obtained a nice red ISH signal.
As the primary antibody for ICC is produced in sheep, I had to previously
elute the anti-DIG antibody, which is also produced in sheep. I use a
standard elution protocol with 100 mM glycine-HCl pH 2.2. And after my
elution, much to my despair, the red in situ signal was gone !!! Normally
the NovaRed substrate is supposed to resist even dehydration and clearing
in toluene. But even there I've had many surprises yet. About half of the
experiments, my NovaRed signal disappears or is significantly reduced after
ethanol dehydration, clearing and coverslipping. Mostly, I now treat the
NovaRed signal as alcohol-soluble and treat the sections with Crystalmount
before coverslipping.
At first I liked the NovaRed substrate quite a lot, because of its
sensitivity. If your antibodies are correctly titrated, you can even forget
your slides in the NovaRed for 1 hour without getting background. But I no
longer believe in it's "alcohol insolubility", and probably even in its
insolubility in aqueous acid solutions. Has anyone observed this in acid
differentiation of hematoxylin counterstains ???

My recommandation to those doing double stains requiring antibody elution
with acid ... use AEC.


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