I am trying to use 5-10 days cotyledons and stain the nuclei with dapi in a GFP tagged plant, anyone has a trick to make the DAPI penetrate ?
DAPI for plant sample is not a very good choice as it is semi-permeable. You may add 0.2% Triton x100 to the sample before adding DAPI solution and may apply vacuum for 2-5 min. This will enhance the dye penetration.
Hi Hugo how are you ?
Actually, fixing the tissue in 1% formaldehyde for 15 minutes will allow DAPI to efficiently enter the cells, but this will kill almost all of your GFP fluorescence. So I think the best solution for you is to vacuum infiltrate a solution of Hoechst-33342 at 1ug.mL, let incubate your plantlets 30 minutes in the dark and directly image the cotyledons.
I have stained Nicotiana nuclei with diluted DAPI in PBS by sinking overnight the cut material (e.g. a 4 mm2 leaf piece) in microfuge tubes. Then I washed with PBS in the morninng and done vacuum with a syringe to remove the air inside the cells. I have not tried in Arabidopsis but I guess it may work as well.
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