Re: Paraffin processing BCIP/NBT stained embryos


Date sent:      	Mon, 18 Nov 2002 15:51:20 -0600
From:           	"Johnson, Teri" 
Subject:        	Paraffin processing BCIP/NBT stained embryos
To:             	"Histonet (E-mail)" 

> Hi again,
> I have been asked to paraffin process some embryos which have had ISH done on them using BCIP/NBT as the substrate.  How stable is this chromogen for this process?  I suspect some of it will leach out in the alcohols.  I use Pro-Soft (propanol, propylene glycol ether, propylene glycol ester) to 
dehydrate and aliphatic hydrocarbon to clear.  I'm hoping this will help minimize chromogen loss.  Additionally I plan to limit the amount of time in the dehydration stages.

Teri, you may well lose some of the chromagen on processing, it 
seems to be variable. Even when the embryo appears dark blue, on 
sectioning it can be difficult with paraffin wax sections to visualize 
any cellular staining. I tend to do frozen sections on these which 
allows a thicker section to be cut and no loss of chromagen, also I 
find frozens easier with regard to orientation. 
If you have a "spare" embryo with the some, you may want to hold 
one back for frozen sections as a control.
Good luck,
Terry Hacker,
Head of Histopathology and Electron Microscopy
Medical Research Council,
Oxfordshire, OX11 ORD
01235 824521
Fax 01235 834776

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