Is GPF/YFP signal OK after fixation? - Does fixation for immunofluorescent stain cause loss of GPF/YFP signal (Feb/15/2008 )

Hi all,

My cells express YPF and protein of my interest (YFP-ires-GENE) and I plan to immunofluorescent-stain on the cultured cells on the coverslip (YFP signal + another signaling molecule signal). I worry whether or not YFP signal will disappear after fixation either by methanol or paraformaldhyde.

(I normally use methanol)

Please give me some information about this. Thanks!!!

I've had cells that I've transiently transfected -- and my protein has been tagged wit YFP. For an immunofluorescent stain, I've only used paraformaldehyde and never saw a loss of signal. If the cells are exposed to light or the reagents that you're using aren't very good, there maybe a reduction in signal strength.
However, I can't really comment on the results if you were to use methanol.
Sorry I can't be more help.

Thank you! I will try both!!

As long as the pH of paraformaldehyde is around 7.4, it should preserve fluorescence.

Your signal should remain after either methanol or paraformaldhyde. I was trying to find a way to actually quench the GFP signal so I could do a stain in the green channel but was unable to destroy the signal without using denaturing conditions (low pH) which destroyed the epitope on my other desired protein. So no worries for you... your signal should be the same (or even stronger!) after fixation. Just be sure to keep them in the dark at all times!