Hi,
Has any one try to immobilize cell nucleus on dishes or slides?
If you did, how did you do it?
I am assuming that I might be able to do it with coating dishes with material having opposite charge to nucleus membrane charge, but not sure how it would work.
Please let me know if you have tried it.
Thankss!
Hello Bumjun.
You could try any plant-derived lectins such as Con A, WGA and PHA at 2~5ug/ml lectin/PBS.
Drop 10ul lectin solution on glass slide or coverslip (or cover the whole dish with lectin solution) for 1h without disturbance) under lamina flow, dust-free with cover (or dish lid).
Wash them in ddH2O (dust-free & sterile) three times gently without touching them to remove salt component (tends to crystallize, hindering nuclei attachment).
Air-dry completely in the lamina flow.
Wash your nuclei solution in ddH2O three times at gentle speed to prevent from sticking each other.
Take nuclei solution on the lectin-coated surfaces.
Leave them for 30 min or so.
Glycoproteins on the nuclear envelope membranes will bind to the lectin molecules, getting pizza-shaped nuclei.
Wash with ddH2O gently with buffer of your choice.
(Hope lectin would not be a barrier in your next procedures, You did not give any info).
Good luck.
Thank you so much for your advice!! I will try that.
There is one thing that I am wondering. You have mentioned that I will be getting pizza-shaped nuclei. What does that exactly mean? Nuclei in general looks circular and little bit flat. Did you meant that nuclei will be flattened out more once it binds to lectin molecules?
I am also wondering if you have any articles that I can refer to.
감사합니다 교수님!
Hi Bumjun.
That was a kind of firm binding nuclei to the coverslip after centrifugation to do more tough process like Triton X-100 extraction, removal of proteins, localization of a gene by FISH or BrdU-incorporation procedure.
However, you may have rather flattened nuclei instead of round balls. Use lower lectin, need to negotiate the concentrations to get maximum number of nuclei and not flattend too much. Have a look though it's about cells, not nuclei. But you will get some idea about what would look like.
https://pdfs.semanticscholar.org/81fb/82e35fe84d604861158ce7cc2ecd682545db.pdf
Best wishes and all the blossoms!!!
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