From:Ronnie Houston <>

Just a quick addendum about dehydrating sections reacted with Nitro BT;

Nitro BT forms a diformazan when reacted with a dehydrogenase. The red component is soluble in alcohols/xylene and will be removed. This is not important in routine histochemistry, but will lead to conflicting results if any quantitation of enzyme activity is being considered. 

Ronnie Houston
Cytochemistry & Molecular Pathology
Texas Scottish Rite Hospital for Children
2222 Welborn Street
Dallas, TX 75219
(214) 559 7744
(214) 559 7768 - fax

>>> Lee & Peggy Wenk <> 07/19/01 06:56PM >>>
Just a few thoughts. 

1. Any chance it is NOT fresh frozen tissue? In other words, it's 
fixed frozen, or worse, fixed processed? This is an enzyme 
destroyed by fixation.

2. Is the pH of the TRIS buffer correct? Sometimes, even with
adding the correct amount of reagents, the pH is not 7.4, due to 
the d. water having some contaminations (which it shouldn't but 
sometimes does), or the HCl is old, so not as acidic.

If not correct, adjust with dilute HCl  or more TRIS. (I think 
dilute Sodium hydroxide could also be used to raise the pH.)

3. Are the amount of NBT and NADPH correct? For 10 mL of
buffer, add 0.01 g NBT and 0.004 g NADPH. Sheehan lists
it in mg, so sometimes it gets converted to grams incorrectly.

4. Is it NADH/NADPH? There is a NAD and a NADP, without
the Hydrogen, that can be bought. You need the type with the
hydrogen, which will get released by the patient's enzyme. The
hydrogen will the bind to the NBT, and reduce the NBT to a 
blue formazan compound.

My top four reasons why it won't work. Let me know if
any of these work. I'm curious.

By the way, for when your procedure starts working - we do 
not counterstain. Also, after rinsing in water in step 5, we run it 
up through alcohols and xylene, and then coverslip with synthetic 
mounting media. We do not see any loss of intensity, and the 
pathologist love it, as resolution is better and the slides can be 
stored easier and last forever, compared with aq. mounting media.

Good luck to you and your colleague.

Peggy A. Wenk, HTL(ASCP)
William Beaumont Hospital
Royal Oak, MI 48073

----- Original Message ----- 
To: <>
Sent: Thursday, July 19, 2001 3:55 PM
Subject: NADPH

> Help! A co-worker is trying to do NADPH for the first time and is
> getting no reaction. He is doing procedure from 2nd edition of Sheehan

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