Living cell GFP imaging - (Jan/19/2008 )
Hi dear brothers & sisters!
these days I am doing a subcellular localization assay of a GFP fusion protein using transgenic Arabidopsis. the fusion is predicted to be nuclear localized; however, in my previous experiment, no cell was found with nucleus, which made my experiment meaningless. I wonder whether it is a common phenomena that only few cells of hypocotyl or root tip contain visible nuclei? Another question, If the subcellular localization of the fusion is light-sensitive, is it correct to pretreat the etiolated tissue with formaldehyde before observing the GFP fluoresence? Someone did so but some other did not, depending on different documents. What should I do so?any suggestion would be greatly appreciated .
Thanks in advance!
Paul
On live mammalian cells, we donot use formaldehyde if we wish to visualize fluorescence. If you can fix with formaldehyde (they are not live anymore), you can get good pics.
But will the gfp protein retain its ability to emit fluorescence after formaldehyde fixation?
Yes it does. It might loose some intensity, but should be OK if expressed in adequate amounts.
GFP fluorescence will be retained after fixation but try to have the pH of the fixative at pH 7.4. Different pH, you can have problems with fluorescence.
GFP fluorescence will be retained after fixation but try to have the pH of the fixative at pH 7.4. Different pH, you can have problems with fluorescence.
Thanks a lot!
GFP fluorescence will be retained after fixation but try to have the pH of the fixative at pH 7.4. Different pH, you can have problems with fluorescence.
Thanks a lot!