NBT/BCIP in situ detection problem

Paul Klosen klosen at neurochem.u-strasbg.fr
Mon Oct 23 04:27:02 EST 2000



Paul Grimm wrote:
> 
> Is it possible that the mounting medium or technique is different and affecting
> the NBT/BCIP deposits? I used glycerol gel to mount the cover slip when I was
> using NBT/BCIP for in situ's. Sometimes a batch of glycerol gel would not work
> as well as another.
> Suggest that you try temporarily mounting one of your slides with water or
> saline and a coverslip to see how the deposit looks under the microscope before
> formal mounting.

We have done that, and we observed the granular appearance even when the
slides are mounted in buffer or glycerol containing media. We always
test the appearance of the development this way to select the slides
that we process further for in isut/ICC double labeling. Also, this
helps us decide whether the signal is strong enough to "clean" the
reaction with ethanol to obtain a blue label, which contrasts better
with the red ICC than the dark violet original label. Our final mounting
medium is the watersoluble "Crystal mount". Upon air-drying this medium
increases its refractive index and forms a film over the sections which
allows a standard Eukitt mounting of the coverslip and a perfectly
transparent preparation. We have never seen any difference in the
appearance of the precipitate in the buffer and after mounting with
"Crystal mount".

> The other issue is whether you use polyvinyl alcohol in your NBT development. I
> think PVA can give a more "granular" appearance.

Good point ! Indeed, we normally use PVA to increase sensitivity and
obtain a crispier precipitate. Thus our first reaction was to
incriminate the PVA. But we still use the same batch (and even the same
container) than previously, and we have tested without PVA, without
improving the result :o(

Thanks

Paul
-- 
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. 03.88.35.85.04  Fax. 03.88.24.04.61  klosen at neurochem.u-strasbg.fr






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