Hi Julie,
Thanks for the replies :)
I did have a search on Pubmed before I started all this but was not able to
find either any consistent protocols or any decent photographs. I would be
very grateful if you could sen d me your protocol for NADPH-d staining. The
brainstems are very old formalin fixed tissue which I have cryoprotected and
sectioned to 50um. They have been stored in the cold in PBS since then -
about a month. One more question i have is that, now I have stained them or
maybe overstained them, is there a way of removing the stain that is there,
eg, with alcohol or something like that.

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 4:09 PM
Subject: RE: NADPH-diaphorase


>
> Anila-
>
> 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?
>
> Julie
>
> -----Original Message-----
> From: Anila Syed
> To: Julie Maier
> Cc: histonet@pathology.swmed.edu
> 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
> 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 a