Hi there. I am performing some life imaging experiments and would like to look at the nucleus using either Dapi or Hoechst. I am wondering if anyone could share a protocol. My understanding is that while Dapi is more photostable than Hoechst, it can also be lighter and hard to see. I have tried a few experiments and cannot see the DAPI at all. Wondering if I need to switch over.
DAPI is not typically used in live cell imaging because it is not very good at crossing intact cell membranes. Hoescht is a much better alternative. Phototoxicity is more likely to be an issue than photobleaching of either of these dyes.
Hoescht is better as it is more stable; You can try once- use hoescht solution in the imaging media.
Bethany Unger
Hoechst33342 is cell permeable and usually used in live cell imaging. However for long term imaging, the UV laser illumination creates a lot of photo-toxicity.
Its recommended hence, to use far-red DNA dyes like To-pro3. In my hands, Hoechst staining apart from photo-toxicity also affected my samples as their replication was affected....just a heads up, even though it may not be an immediate issue.
Beware that some of other Hoechst family dyes are cell impermeable like Hoechst 33358.
Hope this helps
Best
Aparajita