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
-peixumol-
QUOTE (peixumol @ Jan 19 2008, 08:18 AM)
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
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
Are you using confocal to visualize your GFP? If not, then I would highly recommend that you try it. I think with the confocal you should be able to easily see nuclei in most cells of the root tip. The hypocotyl might be slightly more difficult because of the chlorophyll, but in the root tip it is usually very easy to see. Also I assume that one could DAPI stain root tips, but I've never tried this myself.
-smu2-