From:Anila Syed <>

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 sense to someone,

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