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Immunocytochemistry Forum Immunocytochemistry Forum. Discuss methods and immunocytochemistry techniques for cell suspension, cytospin, or cultured cells.


storage of PFA fixed cells

storage of PFA fixed cells - Immunocytochemistry Forum

storage of PFA fixed cells - Immunocytochemistry Forum. Discuss methods and immunocytochemistry techniques for cell suspension, cytospin, or cultured cells.


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Old 11-10-2009, 08:44 AM
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Default storage of PFA fixed cells



Hi,

I'm culturing cortical neurons and I'm looking for a protocol to store PFA fixed cells so that I don't have to stain them all in one go. How long can you store PFA fixed cells?
I would great if anyone could help me out here!

Thanks a lot for your help!!!
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Old 11-10-2009, 02:46 PM
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Default Re: storage of PFA fixed cells

We often keep PFA fixed cells for 1-2 weeks. Fix for 4-8hrs in 4% PFA at 4C, wash with PBS and then store the cells in the fridge covered with PBS.

That said, we almost always to the staining immediately after fixing. I have no idea how long you can store the cells without fixing before staining will be compromised.

Bryan
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Old 11-12-2009, 01:09 AM
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Default Re: storage of PFA fixed cells

i guess it depends on what antigen you are staining for. i have stored fixed cells in PBS at 4 degrees for weeks and the immuno still turned out very well.
remember to make sure you keep wells/containers air0tight...parafilm or cling wrap and check to make sure it doesn't dry out! I have done that by accident...dried out cells don't look very good and it feels like a huge disappointment!
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Old 11-12-2009, 08:29 AM
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Default Re: storage of PFA fixed cells

Thanks a lot for your input! Next time I will give it a try. Do you add any preservative like Sodiumazide to the PBS to prevent bacterial growth?

Thanks again!
Gernot
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Old 11-12-2009, 03:37 PM
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Default Re: storage of PFA fixed cells

I've never used a preservative; PBS doesn't contain much bacteria can use, so as long as you use clean techniques and keep it in the fridge, things should be fine.

I forgot to mention the sealing with parafilm - that is very important!

Bryan
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Old 11-13-2009, 01:44 AM
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Default Re: storage of PFA fixed cells

Oh and I have actually used sodium azide as an endogenous peroxide block for the 2nd stain in double immunos.......just another use for azide...but if you don't need to, best to avoid this uber-toxic chemical
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