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immunofluorescence for HUVEC


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#1 smilealie

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Posted 05 January 2010 - 07:11 PM

hello everybody,

i am new to cell culture.my boss told to do immunofluorescence but i am facing problem in fixing the HUVEC cells.i used normal procedure done by others in paper.but when i going to fix the cells the cells are shrinking.so kindly help me how to get rid of this.
Is anybody having detail protocol for immunofluorescence for HUVEC cells.


thanks in advance,

regards,
alie

#2 bob1

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Posted 06 January 2010 - 03:13 PM

Which proceedure did you use? Methanol/acetone or paraformaldehdye? If the former, try using 2% formaldehyde solution for 10 minutes. Otherwise, did you re-hydrate your cells after fixing in methanol? If not, you need to by serially increasing the concentration of water in the fix solutions e.g. from 100% methanol, add 80%, 60%, 40%, 20% methanol and finally plain water or PBS for 5 minutes each.

#3 smilealie

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Posted 07 January 2010 - 06:10 PM

thanks bob..

as soon as i add acteone/methanol on coverslip the cells started to shrink and looses its shape.so could not proceed further.
if u have any good paper where they have used HUVEC CELLS experiment...

#4 bob1

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Posted 10 January 2010 - 04:03 PM

the methanol/acetone shrinks the cells by dehydration. Use formaledhyde if you need to preserve cell shape.




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