Hi, Julie,

Many thanks for your response,
I have just done a test run on some sections and the results were horrible.
They were completely brown!!! I haven't cleared them yet, but I am not very
hopeful. I though they were supposed to be blue ?? No-one has any photos of
any do they?

Many Thanks,

Anila
----- Original Message -----
From: Julie Maier <Julie-Maier@mail.omrf.ouhsc.edu>
To: 'Anila Syed ' <syedab@totalise.co.uk>
Cc: <histonet@pathology.swmed.edu>
Sent: Friday, August 03, 2001 1:59 PM
Subject: RE: NADPH-diaphorase


> 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 thi