RE: NADPH-diaphorase

From:Julie Maier <>


You're right, results should give you a nice purple/blue section.  I don't
have any pictures to send you, but if you get on MEDLINE or PUBMED and look
up NADPH-diaphorase there should be some pictures in various journal
publications.  If you would like, I could send you a copy of my protocol.
You may want to try incubating a little less (sections could be so purple
that they look brown).  Also, how are your brainstems treated?


-----Original Message-----
From: Anila Syed
To: Julie Maier
Sent: 8/3/01 8:46 AM
Subject: Re: NADPH-diaphorase

Hi, Julie,

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

Many Thanks,

----- Original Message -----
From: Julie Maier <>
To: 'Anila Syed ' <>
Cc: <>
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
> were using 30um cryosections which were placed (floated) in diaphorase
> solution immediately after being cut (no cold incubation), incubated 1
> at 37C, then rinsed in PBS prior to floating onto slides.  Our PBS was
> 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
> 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
> Tris buffer is at. Can I assume that the actual buffer is not
> 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
> 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 t

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