[Histonet] queries re freezing PFA, and storing tissue after fixation

From:"PALMER Jason (SVHM)"

Hi histonetters.

I know there has been quite a lot written on histonet about how formaldehyde fixatives work, and comparisons between PFA vs buffered formalin solutions for fixation etc, and I have read many of these, but there are still a couple of issues relevant to my lab that I'm not quite sure about.

I work in a research setting and the tissues we process are mostly mouse or rat, and quite small, I think - say 3 x 6 mm on average, or something like that. We do lots of immunohistochemistry, and normally fix our samples in 4% PFA / PBS for 24 hrs at 4 degrees C. There are a couple of practices in our lab that worry me a little, and I'm not sure who instigated them and nobody can give me good explanations why they are acceptable. One is the use of PFA that has been frozen. It is made up in the usual way, aliqoutted and put into the -20 c freezer on the same day, stored there, and thawed out on the day (or day before) of use. Making PFA in one biggish lot and freezing aliquots makes sense in terms of saving time and effort, but is the PFA going to remain "stable" and not start to repolymerise over what could be weeks, or even months, in the -20 c freezer?? Personally, I'm not even convinced that freshly made and used PFA is better than 10% buffered formalin for our immuno work, on the whole, though that's another issue...

Secondly, it is often the case here that after fixation, tissues are stored in PBS (in the 4 degree C fridge) for what may be weeks rather than days before processing. I personally haven't notices any difference in the immunostaining seen with these samples, compared with samples processed immediately or within days, for a number of antigens, but I still feel that storage for weeks in PBS is not a good idea. From what I've read, formaldehyde fixation for the time we use (24 hrs or sometimes even a little less) is reversible over time, in PBS especially. I guess an alternative is to store in 70% ethanol, but again I can't quite help wondering if this might not be conducive to good and consistent immunostaining.

Any thoughts much appreciated,


Jason Palmer
Bernard O'Brien Institute of Microsurgery 
42 Fitzroy St, Fitzroy Victoria 3065
tel +61 3 9288 4018
fax +61 3 9416 0926
email: palmerj@svhm.org.au

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