Antibody elution and NovaRed

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From:Paul Klosen <>
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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.

                                                                        (o -) O
Paul Klosen, PhD
CNRS UMR 7518 Neurobiologie des Fonctions Rythmiques et Saisonnieres
Universite Louis Pasteur  12, rue de l'Universite
F-67000 Strasbourg, FRANCE
Tel.  Fax.

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