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

[Kathleen Spencer]

We store rat brains in 20% sucrose in the fridge until we take them out  
for cryosectioing. Our animals are perfused with very fresh 4% PFA  
only.

Kathleen


On Jan 30, 2005, at 11:10 PM, PALMER Jason (SVHM) wrote:

>
> 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
>
>
> Jason Palmer
> Bernard O'Brien Institute of Microsurgery
> 42 Fitzroy St, Fitzroy Victoria 3065
> Australia
> tel +61 3 9288 4018
> fax +61 3 9416 0926
> email: palmerj <@t> svhm.org.au
>
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