Anila-

I did NADPH diaphorase for two years staining for nNOS in rat brain PVN.  We
were using 30um cryosections which were placed (floated) in diaphorase
solution immediately after being cut (no cold incubation), incubated 1 hour
at 37C, then rinsed in PBS prior to floating onto slides.  Our PBS was pH
7.4.  Hope this helps!

Julie

-----Original Message-----
From: Anila Syed
To: Histonet
Sent: 8/3/01 5:21 AM
Subject: NADPH-diaphorase

Apologies if this question has been asked before,

I am using the NADPH-d stain for the first time, staining free floating
50um
sections of brainstem.
I have been given a protocol for muscle and am filling in the details
from
websites and asking around.
I hope that a kind individual can answer these questions that i have:

Some ppl use Tris buffer and some use PBS and it doesn't say what pH the
Tris buffer is at. Can I assume that the actual buffer is not important?
But
the pH is?

Also, in soem protocols, there is a cold incubation in buffer before
hand
and then a 1.5 hour incubation at 37 deg C in NADPH and nitroblue
tetrazolium and in others there isn't. What is the reason for incubating
in
the cold? My sections are coming straight form the fridge in any case.
Would
I have to do this incubation? Is it just to bring it to the right pH?
Wouldn't the temperature affect the pH anyway?

Hope this all makes