Hello Malo, it is possible if you have a strong laser for excitation. Because DNA staining is pretty strong, it is usually sufficient. Still, it will bleach your sample quite fast.

I use it routinely.

Dear Malo

Most of us routinely use Hoechst 33342 for immunofluorescence and excite it with a 405 laser. It works perfectly fine. Just make sure you have the emission filters for Dapi/Hoechst in your microscope or else, it will be quite hard to see.

Good Luck

Thank you all for your answer,

Aparajita Lahree No problem I have this filter!

Any concern about toxicity ?

Regards,

Malo

If you are using Hoechst 33342 in live samples, yes there is a chance of toxicity, but for fixed samples its not a concern.

If the imaging window is small, go ahead but if you plan on long term live imaging I recommend other functional DNA dyes that are less toxic to live cells such as fluorescent protein labelled histones (need to transfected before into cells) or SiR-DNA.

Good luck

Yes I want to use it on live samples, planning around 1hrs/2hrs imaging. I'll try like this, I just want to perform easy staining, it's not the main information of my experiment.

Any advice for protocol ? 20mn TA then washing (using on coated cells in ibidi/labtek)

Thanks again,

Malo

It'll work, but not efficiently. Poor for microscopy, passably for flow cytometry. A better choice is Hoechst 34580, which has a higher excitation peak that is easily excitable at 405 nm.

Hello Malo

I think 20 min staining with Hoechst33342 in culture media would be fine, but since its live, there is a possibility that dye incorporation may not be uniform and you may need to use high laser power for visualization. This in turn would be photo-toxic for cells. Just to have a wider emission range you can use Dapi, if there is nothing in the green channel in your system. Since Dapi has a long emission coverage in wavelength, you can catch light from a broader filter gap setting. Both Hoechst and Dapi excitation at 405 is possible but not greatly efficient as Jason mentioned.

If at all it is possible for you, try to use Far red DNA dyes in the future, as they are less photo-toxic for cells and some work quite well in terms of incorportion.

Good Luck

Some news :

It works really well for me with Hoechst under 405 excitation, with very few toxicity on one hour visualization. I used low laser power (can't remember how but less than 1%).

Note that I work on confocal zeiss LSM 780.

Thanks all for your answer, find attached a picture of my staining on Hela Cells:

Have a good day

Looks great Malo. Congrats :-)

Aparajita Lahree Hey, I want to stain nucleus of algae with Hoechst 33342 but problem in permeability, i think coz of cell wall. So can u help what can i do?