I tried fixing the cells, treatment with 2 % SDS but the dye is not goin inside the cells.
Hi Neha,
Do you mean micro-algal cells?
DAPI staining might depend on
1)concentration/dilution of the dye 2) Incubation conditions (Buffer/pH/incubation time/ temparature dependant)
You may check these papers for further details
1. http://www.tau.ac.il/lifesci/departments/zoology/members/benayahu/documents/253-Zurel-final.pdf
2. http://www.nature.com/protocolexchange/protocols/4075
http://www.tau.ac.il/lifesci/departments/zoology/members/benayahu/documents/253-Zurel-final.pdf
Hii Shreyas,
Thanks for your help. I tried using 0.3 mg/ml and 0.1 mg/ml DAPI in PBS (pH 7.2) and my incubation time of about 15 min. Dye is getting accumulated at the periphery of the microalgal cells.
You might try to add 0.1-2 % dimethyl sulphoxide. A further increase of the incubation time (up to overnight) might also help. Good luck!
Thanks Alexei , I did stain overnight but the stain is getting accumulated on the periphery of the cells. Ill try using DMSO.
Heey, I was wondering if you'd figured out the issue regarding the permeability of the stain. I am facing the same issue with DAPI and Hoechst stains.
Which one is better for staining algal cells DAPI or Hoechst stains ?
I had no problem with Euglena and Chlorella cells when fixed or dead, by doing ethanol fixation and incubating with DAPI. For Chlorella vulgaris, for instance, if you want to stain both nucleic acid (blue) and polyphosphate (yellow), I used 100 µg/mL DAPI 10 min incubation following Ethanol 1:1 fixation. For Euglena gracilis, 2 µM of DAPI gave good results after a similar incubation. For Chromochloris I found that 0.2 µg/mL 10 minutes in modified Acetate medium worked well. If you want to stain live cells, you will need permeabilization (I have not tried this). I have been using it in either fixed/permeabilized cells (with ethanol) or as a membrane integrity dead cell staining.
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