Fixing the Endothelial Cells - (May/23/2007 )

Hi every one,
Does any one here know how to fix endothelial cell monolayers for immunofuorescence microscopy? I used 2% paraformaldehyde in DPBS but it didn't work very well. The cells looked very weird after the fixation.
I would appreciate your help

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What do you mean by 'weird'?

May be they look weird because they are dead.

We use 4% PFA in for fixing cells.

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By weird I mean they had started to detach from the glass slides after I fixed them.
Do you prepare your fixative fresh every day? Do you fix it at 37 or cold?
Thanks

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Did you make PFA in PBS with calcium and magnesium? This can help.

Also coat the wells with poly lysine or ornitthine before you seed cells and then fix them.

We make PFA every 2-4 weeks and store in 4C. But the fixing is done in RT.

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Thanks a lot scolix. I made PFA in PBS with calcium and magnesium to help them not detach. I also treat the slides with Collagen I.
Is it OK to freeze the PFA and thaw it once we want to use it?

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We leave the cells in PFA for 5-10 min at RT. Did you try altering time required for fixing to prevent cells floating?

I never froze PFA, so I have no idea. But You could make up 8% PFa and store it in 4C and use it for upto 2 months.
ofcourse you need to dilute it before using.

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Thanks a lot Scolix. I fixed them for 15 minutes. It might be long, you are right. I need to fix them for 5-10 minutes and see how it affects the cells.
Thanks again

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We did make 15% stock, freeze aliquots of it, then thaw and dilute to the usage concentration (3%) when necessary and it works fine.

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