Hi thanks,
for your advise, I decided to start making PFA fresh every week.
After all is not that much work and if results are better it pays off the effort.

Is it important to filter the PFA solution?

Macedo

i dont filter but i do microwave (stand well back!!) to help it dissolve

dom

Microwave may not be very good to PFA, it becomes FA instead of paraFA, I think. Just use warm (not boiled) PBS should be fine.

Hello,
Paraformaldehyde is a chain of formaldehyde molecules. If you want to bring it into solution, you must warm it up until around 60 °C. By doing so, you open the chain and the individual formaldehyde molecules are set free in the solution. Of course you can add also phosphates to bring the solution to the requested pH. Microwaving is another method of breaking up the chain.
The advantage of paraformaldehyde is that you are sure of the formaldehyde concentration in your solution. When you start with a liquid (formaline: +/- 37 %) you are not so sure of your formaldehyde concentration. It can be 37 % but it can also be less because if the concentrated solution (formaline) is already staying for some time on the shelf, there will be other products starting to develop in the solution (paraformaldehyde, formic acid...). The formaldehyde concentration will decrease in time and the other products in the solution can have a negative influence on your reaction.
Frank

thanks frank

We always use 4% paraformaldehyde. I warm the suspension and then add 1N NaOH to adjust pH (Say 100 microl to 100 ml PFA solution). For fixation of the cells pH of the solution is very important and it should be 7 - 7.4
I give 2-3 quick washes with 1X PBS and add PFA mentained at 370C. it helps in mentaining the morphology of the cells.

regards

That brings back my old question - how do U know if cells are fixed or not?

May be we should be careful with that. Fumes of formaldehyde are toxic and formaldehyde is also a carcinogen.