Hello,
I am trying to arrange a protocol for immunostaining of Nicotiana benthamiana leaves. We look into our samples using confocal microscope but despite we have strong signal compare to the control, it was dificult to see any nuclei, what we saw instead were chloroplasts.
Any alternative to PBS Triton DAPI for detecting mesophyll nuclei?
Thank you
Hello,
Since chloroplasts contain DNA, they will be readilly stained with minor groove binding DAPI. You may obtain picture like this: Chapter Development-Dependent Changes in the Amount and Structural O...
Triton X-100 and Tween-20 are permeabilizing membranes non-selectively, so probably you will need to optimise concentration or select other detergent.
Thank you for your answer.
I don't know why nuclei do not stain much more than chloroplast.
Could it be since I am using sections of 7 um? Maybe that's why I do not see a nucleus in each mesophyll cell.
I will try to adjust the thickness and detergent concentration.
Could you kindly attach brightfield image of the same section? I think you may first omit the sections to just verify the appropriate detergent concentration and nuclei staining.
I have torn apart one yong tomato leaf, removed upper side and incubated it in DAPI (~10ug/ml in 0.9% w/v NaCl) for 30minutes. I can observe bright nuclei-shaped and sized objects in mesophyll cell mass.
Thank you for your answers, in my case that is difficult because I need to immunodetect some proteins in the leaves apart from visualizing the nuclei.
It's OK, my point that nuclei might disappear because cut thickness is relatively small. Second point is that if you are already running ICH, so why not use the hantibody against istones or PCNA or anything else? E.g. DAPI for plastids, two other channels for nuclear-specific protein and your protein of interest?
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