I want to examination tumor cell apoptosis. Tumor cells were fixed by methanol.
I think DAPI stain will be perfect for apoptosis assay/examination.
even i would like to say that if don't have DAPI or Hoechst 33258. you can go with EtBr/Acridine orange stain (1:1). since we carry out apoptosis assay against cancer cell line and we got good result.
If you want to look at caspase3/7 dependent apoptosis, you could also use a substrate called NucView488. It will stain the nuclei of apoptotic cells (where caspase3 or 7 are active). You can do a counterstaining with Hoechst. There is no need to fix the cells for that purpose.
Good luck,
Cindy
Hi, in practice there is very little difference. Dapi is slightly more photostable than Hoechst, but you would only notice with very long exposure times. Like others above suggested, it is probably wise to also stain for some earlier markers of apoptosis, as you would miss a lot if you only look at pycnotic nuclei.
Thank U Cindy Körner, your suggestion is very usuful. I will conduct a counterstaining by Hoechst without fixation.
Dear Hendrik Gremmels, Do you know some earlier markers of apoptosis for fluorescent microscope examination? Thank you.
Dear Ruixian,
Nuclear condensation and shrinkage is the characteristics features of apoptosis. For condensation and shrinkage you can used either hoechst or DAPI. Both equally work. For the best microscopic image for the same, you have to fixed the cells in ethanol (90% chilled for overnight) or fixed the cells in 4% para-formaldehyde (for 15 min at RT). After staining you can mount you cells with glycerol or any other mounting media and store it at 4 degree temp.
For earlier apoptosis marker determination, Annexin-V is the only and widely accepted marker which stain early apoptotic cells.
Regards
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