For GFP localization study by confocal microscopy, I need to prepare a permanent slide of the protoplast samples which are prepared from the agroinfiltrated leaves of Nicotiana benthamiana. Is this possible or protoplasts will die ? Is there any protocol available for this.
Thanks
Dear Panchsheela Nogia
Pls. find the attached files, may help in your research, good luck
http://www.nature.com/nprot/journal/v2/n7/full/nprot.2007.199.html
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2896036/
http://molbio.mgh.harvard.edu/sheenweb/protocols/Atproto02RL12.pdf
http://molbio.mgh.harvard.edu/sheenweb/protocols/AtprotoRL04.pdf
http://dare.uva.nl/document/2/111459
http://www.plantcell.org/content/23/3/1153.full
https://www.researchgate.net/publication/237065360_Laboratory_Protocols_in_Applied_Life_Sciences_by_Prakash_S_Bisen
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203094/
https://www.researchgate.net/publication/51901937_The_Use_of_Agroinfiltration_for_Transient_Expression_of_Plant_Resistance_and_Fungal_Effector_Proteins_in_Nicotiana_benthamiana_Leaves
http://www.bio-protocol.org/e1412
https://www.mpipz.mpg.de/25781/Mathur_PEG_transformation.pdf
http://www.saps.org.uk/secondary/teaching-resources/133-protoplast-isolation
Regards
Prof. Houda Kawas
Book Laboratory Protocols in Applied Life Sciences by Prakash S. Bisen
Article The Use of Agroinfiltration for Transient Expression of Plan...
Thank you, Prof. Houda Kawas but we need to prepare slides which can be stored for few days while all the information available online is about preparation of a temporary mount which has to be visualized immediately.
You can put them in a refrigerator to prolong their life. Maybe just for a few days they will stay alive. You can use a vital dye to assess whether they are still alive after a few days in the refrigerator. It is just impossible to prepare a really permanent slide with living cells, let alone protoplasts.
Dear Panchsheela Nogia
Could you have a look at the troubleshooting in the first article.
Regards
Houda
Actually ,we have to carry our slides to other lab, because we don't have confocal facility here so it has become a problem that how to store those slides so that there should be alive protoplast with enough GFP fluorescence.
Hi Panchsheela,,
1. Protoplasts are plant cells without cell walls. They are very fragile. They can burst like air bubles if you don't handle them carefully. They can burst due to violent shaking, medium drying up or in wrong solution/medium (protoplasts are osmotically sensitive, as in a suspension medium hypotonic to that of its protoplasm, water enters the cells by osmosis. This inflow is prevented by the rigid cell wall.)
2. People have mount 'plant tissues' for observing GFP-tagged sub-cellular organelles. However, I am unsure it is ok to mount protoplasts on a slide and cover them with a lid. This may crash protoplasts. In addition, the protoplast solution can dry up during transport, which will damage the protoplasts as well.
I attached this paper for you. Please see the Subcellular localization section at Methods, about how to use protoplast for their confocal imaging (see attachment). It seems that they did not mount the protoplasts. The keep the protoplasts in wells.
3. To carry your protoplasts to other locations, you can keep your protoplasts (in solution of course) in a sterile dish or a FalconTM tube (on/in the ice). I prefer FalconTM tube since it avoid spill-out while doing transfer, compared to using a dish. Once you arrive the room with confocal microscope, you can do the rest preparation work there, including mount them (if you must).
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