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Freezing cells after fix & perm and also a BrdU question


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4 replies to this topic

#1 cardosopedro

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Posted 02 June 2009 - 07:48 AM

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 -20 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 -20 C?


Thanks.

#2 illuminated

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Posted 08 June 2009 - 06:48 AM

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).

#3 cardosopedro

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Posted 15 June 2009 - 02:59 AM

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.

#4 illuminated

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Posted 23 July 2009 - 06:47 AM

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

#5 Brachya

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Posted 04 March 2010 - 08:43 AM

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.




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