Freezing cells after fix & perm and also a BrdU question - (Jun/02/2009 )
This is my 1st post here, although I use protocol-online a lot.
I have two questions:
1) I want to fix, permeabilize and then freeze my cells in order to immunostain them in another day. I saw that this is possible by adding 90% methanol and then storing the cells at -20º C. Is this feasible?
2) Furthermore, one of the stainings that I'm doing is BrdU. Is it correct to expose cells to BrdU, fix them, and store in 90% methanol at -20º C?
Why do you want to freeze the cells for BrdU staining? I usually fix them in 70% ethanol and put them into the fridge where they can stay for at least one week before staining (actually even longer).
Thanks for your answer.
I don't want to freeze cells specifically to to BrdU staining. I was wondering if it is correct to incubate the cells with BrdU, fix them and then store them to do the rest of the staining in another day.
Yes, sure, that's exactly what I meant. BrdU incubation, ethanol fixation, storage in the fridge at +4.
You may stain for Brdu or DNA dyes (like PI) after EtOH or MeOH fixation and long storage at -20 or 4 C. However, I'm not sure this fixation method will allow staining proteins later.