My goi is located in the nucleus and I cloned it into a YFP vector. The screening at the konfocal was successful and now I want to take a closer look at the nuclei.
Dear Snow Nie, thank you very much for the suggestion and the article! I just send an e-mail to Creatice Bioarray...
Best regards, Judith
Dear Judith, for the living cells you can use this dye what is relatively cell-permeable. https://de.wikipedia.org/wiki/Hoechst_33342
Dapi is less suitable for this case.
My best wishes!
Hi Judith:
You can stain with 1 ug/ ml DAPI for 5 min , then wash 3 times 10 minutes each. Alternatively you can use DRAQ5, dyes far red to avoid interference with YFP.
Thank you so much for your help, dear Taras and dear Fernando! First I will try it with DAPI...
DAPI is good for fixing cells. Moreover, if you used real protoplasts, every washing is potential way to damage of the cells, because protoplasts is too sensitive to any centrifugaltion washing.
For the Hoechst, you can add directly to medium and do not need to wash, by using living cells.
@Taras: Ah okay! Then I will switch to Hoechst! Luckily for me: we have Hoechst! What do you mean, which concentration is the best one?
Dear Judith,
approximately 1 mg/l final concentration should work well.
Best wishes!
Hi,
in the meantime I tried to colour my protoplasts with Hoechst33342 (1 mg/ml). Unfortunately unsuccessful... Even next day I couldn´t see any coloured protoplast. Should I add the dye before the PEG / DNA plasmid incubation step? Make it sense to use a low concentration of Tween?
Thanks a lot and I wish all a nice weekend!
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