4 replies to this topic

[cardosopedro]

Hi!
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?


Thanks.

[illuminated]

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).

[cardosopedro]

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.

[illuminated]

Yes, sure, that's exactly what I meant. BrdU incubation, ethanol fixation, storage in the fridge at +4.

[Brachya]

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.