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DAPI/Hoechst staining live yeast cellsHey, I am looking for a protocol to stain

DAPI yeast cerevisiae live imaging Hoechst

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#1 Alfahelix

Alfahelix

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Posted 06 July 2014 - 02:33 AM

Hey,

I am looking for a protocol to stain live yeast cells with DAPI or Hoechst In 1x PBS or 1x PBS with 2 % glucose.

 

So far I have tried using 1 μl of DAPI [1 mg/ml] in 1 ml cell suspension and incubated for 10 min at RT. Washed 3x times prior to imaging. Weirdly enough, staining in 1x PBS with 2 % glucose gave better results than just using PBS. However, the cells were over stained with dots appearing at cell periphery. What

 

For the Hoescht staining I have problems with the whole cell being stained and not being able to get a nice nuclear staining. Here I have tried various concentrations in the range of 2.5-10.5 μg/ml but I have not figured out any pattern.

 

So, which factor(s) should I improve to get a nicer staining; concentration or incubation time?

Also, which staining percentage can one expect for Hoechst staining?  Does Hoechst staining include the nucleolus or just the nucleoplasm?

 

Thank you!







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