Dear Researchers!
The attached images are stably expressing GFP fused target proteins in Brassica rapa young seedlings (Note: leaves were treated with Clearsee solution prior to imaging, to remove autofluorescence). I have three questions to ask.
1. In figure A, what are those strong signalled round shapes (different in numbers) found inside the cells?
2. In figure B, Is it ok to have GFP signals in stomata pores or those are artefacts?
3. In figure C, what are those fluorescence holes on the cell surface?
Can anyone help me in this regard?
Thanks in advance!
I think there are the opposite of each other. The last one is a mix. Does this make any sense to you?.
.... Sdert
Let me tell you that each row (A, B, C) represents different lines and is not related. Also, no matter whether they are opposite to each other. Anyway, My question is meant to ask whether the differential GFP signals represent any of the subcellular organelles?
Q1. I would say chloroplast
Q2. I would say yes,it is ok. (Mesophyll?)
Q3.Maybe artifacts.
Signals: yes they somehow relate to the organelles
.... Sdert
Hi. Thanks for your time. I will repeat the experiments with several different developmental stages and also check for chlorophyll autofluorescence. Coming to your Answers to Q1. If it is the chloroplast, I am expecting more in numbers not very few and also it would present in more cells. However, with Answer 2, I may agree with you. I appreciate your efforts and time. Let you know full results with better interpretation soon. Thanks and have a good time ahead.
Is it living of fixing material? For the organelles location higher magnification (not digital zoom) are required. Please, provide details of the filtres and negative- positive control.
Taras Pasternak
Hi Dr. Taras Pasternak
Those were fixed and bleached (to remove chlorophyll) leaf cells. I used the eGFP filter available with Leica SP8 confocal microscopy which uses 488 nm to excitation and emission spectra lies between 495-545 nm. Unfortunately, in the first attempt, I do not have positive/negative control.
However, recently I tried with live epidermis peel of the same lines. I herewith attaching 2 of those for your analysis.
Here I have more specific questions about whether the GFP tagged proteins are expressed in guard cells (please see the attached photos). Also in chloroplasts?. Same filter sets were used with higher magnification (40x). The GFP signal found only in the inner walls of guard cells?. The second photo attached here is a negative control. Thanks for your time. For chlorophyll autofluroscence (excitation:488 nm; emission 670-700 nm). I hope I am clear with my questions.
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