When reading the multitude of protocols for (W)ISH, most of these mention making PFA fresh from powder, but none that mention simply using commercial pfa solutions (e.g. 16% PFA from EMS, freshly diluted in DEPC-treated PBS to make 4%, kept on ice until use).
Is there a specific reason for preparing home-made PFA solutions instead of using diluted commercial PFA? I wouldn't think there should be much of a difference when either one is prepared fresh for each embryo isolation.
Thanks.
Anecdotally, I once did a comparison of 4% PFA prepared fresh from powder versus freshly diluted from a 37% commercial stock for ISH on rat brain slices (fresh frozen). I found that I had higher background with the slices fixed from the diluted commercial stock, so since then I have been preparing the PFA from powder. Granted the 37% stock was a somewhat older bottle, maybe 1.5 years old so that may have been a factor... (maybe degradation or polymerization?) If you have both powder and liquid stock you could try a side-by-side comparison and see if you have a difference in your system; if not, go with the one that is easier.
Also I guess it may depend on whether the presence of methanol in the commercial stocks is of concern for your preparation.
Thanks. I de- and rehydrate with methanol, so that should be fine. I started wondering about the difference in pfa when my in situs consistently had a low signal/noise ratio with diluted 4% (from 16%), whereas my colleague, whose signal/noise is much better, always makes fresh from powder. But other than anecdotes, I could not find a systematic comparison on Google.
We use to religiously use PFA made from powdered stock. I recently tried using diluted formaldehyde from a 37% commercial stock solution and this worked very well (we're working with snails so perhaps a unique behaviour for our system). Happy with this because I never like playing with the fine PFA dust!
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