From:"" <>

Date sent:      	Thu, 12 Jul 2001 16:43:20 +0100
From:           	Maura Farquharson <>
Subject:        	NBT/BCIP

> Hi folks,
> Several enzyme histochemical methods e.g. for dehydrogenases use NBT as the
> chromogen.At the end of the development of the formazan reaction
> product,several methods recommend fixing for 10-15 mins in 10% formaldehyde
> then dehydating through alcohols,clearing in xylene and mounting.I find
> this loses some of the reacion product and the results are weaker than if I
> aqueous mount.

Allan, I have done a lot of work in the past demonstrating 
dehydrogenases (DT diaphorase actually) using NBT as the 
chromagen.After the post fix as you mention (and counterstaining), 
I would wash and drain the slides then give a quick (5-10 second) 
rinse in acetone to remove any soluble (pink) formazan followed by 
a rapid (final) dehydration in alcohol, clear and mount.The blue to 
blue/black staining was always clear and strong.
Terry Hacker,
Medical Research Council,

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