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From:"Karen D. Larison" <>
Date:Thu, 29 Apr 1999 11:34:36 -0800


The postdocs around here routinedly mount their NBT/BCIP in situs with 
permount, using some of the same approaches suggested below by Dr. Klosen to 
attenuate the loss of signal.  We also embed whole mount embryos with the 
NBT/BCIP signal in plastic.  In this case we dehydrate using a methanol 
dehydration series.  This seems to preserve the signal better, presumably 
because methanol is less lipophilic than ethanol.  We usually don't take these 
measures with sections however, since shortening the time in the ethanol seems 
to work just fine.

Karen at Univesity of Oregon

Date:          Thu, 29 Apr 1999 18:04:20
From:          Paul Klosen <>
Subject:       Re: NBT/BCIP
Cc:            HistoNet <>

This can be done, but should be done with care. Alcohol will extract some
of the NBT/BCIP precipitate, and thus you will lose some sensitivity. Also,
the colour of the precipitate will change from dark purple to blue. I use
this effect to obtain better colour contrast in double labeling with either
DAB or AEC for immunoperoxidase. The alcohol extraction will also greatly
reduce background staining, up to the point where you will have to use
interference contrast to sees some of the section !!!

My advice is to check the label first after mounting in an aqueous
mountant, maybe even take some photos. Then you rinse off the coverslip and
the mounting medium, and dehydrate. A quick dehydration (10-20 dips per
step) will extract just a little of the precipitate. Several authors have
even suggested extended (overnight !!!) rinses to reduce background
staining, but sometimes you will lose faint to medium signals altogether.

An alternative procedure is to use Biomeda's Crystal Mount. Crystal mount
is put on the sections without a coverslip. After drying, it forms a
protective film over the sections, which can then be coverslipped with some
Eukitt or whatever solvent-based mountant you use currently. So far,
Crystal Mount is the only aqueous mounting medium I found that can compete
optically with the synthetic resins after dehydration and clearing. The
down-side of it is that Crystal Mount is prohibitively expensive (at least
IMHO) and that I don't see any reason for this probitive pricing. I am open
to all suggestions about a less pricey alternative to Crystal Mount.
                                   (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

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