GFP imaging - (Nov/28/2007 )
Dear all:
I have recently obtained several lines of transgenic Arabidopsis expressing a GFP-foreign gene fusion protein. As a next step, I want to examine the subcellular localization of the fusion in root tips using a laser scanning confocal microscope. However, even though I have experiences in observing GFP signal in onion epidermal cells, I don’t know how to perform the observation here in planta. Could anybody experienced give me some protocols (the more detailed the better)? Any advices and suggestions will be appreciated.
Thanks a lot.
Yours, Paul
11.28
I have recently obtained several lines of transgenic Arabidopsis expressing a GFP-foreign gene fusion protein. As a next step, I want to examine the subcellular localization of the fusion in root tips using a laser scanning confocal microscope. However, even though I have experiences in observing GFP signal in onion epidermal cells, I don’t know how to perform the observation here in planta. Could anybody experienced give me some protocols (the more detailed the better)? Any advices and suggestions will be appreciated.
Thanks a lot.
Yours, Paul
11.28
We typically grow the seedlings on plates, then at around 4-7 days, set individual seedlings on a slide, add some water and a coverslip. We will usually cut off the top of the root since we're not really interested in that and it makes the slide easier to handle. That is basically all there is to it. Except, be sure to look at wildtype seedlings as well so that you can detect real fluorescence from autofluoresence.
Hi, dear sum2:
Thank you for your kind replysum2. I believe that the method you described here must work well in your lab so that you’d like to share with me. But could you please tell me a few more details about how to make a good resolution? I wonder if root tips, even though are very small by visual observation, would be too thick to be easily observed under microscope?
Thank you once again!