Hi Sebastian!

Have you been able to come up with an efficient protocol that you could share? Which stage are you looking at? I wanted to use mitotracker in adult fly brains...

Cheers,

Catarina

Hey Catarina,

I'm looking at adult fly brains as well. Though it works in principle, I always had some penetration issues with the mitotracker dye. Didn't manage to get satisfying and useable stainings of the whole brain in the end. Works better in other tissues (e.g. muscles). Still I can look for the (unfinished) protocol if you're still interested

That's ok... I got a mito::GFP stock instead to look at mitochondria.

Thanks!

Hi Sebastian,

I was recently trying the mito-tracker dye to stain mitochondria in the adult fly muscle. But I could not find any specific staining pattern. Since you have mentioned that it works well in muscles, could you please share the protocol that you are following for the staining?

Thanks a lot!

Best,

Sid.

Hey, try this protocol taken from doi: 10.1038/s41467-017-00525-4

"MitoSox staining assay. Flies were anesthetized and hemi-thoraces were dissected in cold Drosophila Schneider’s Medium (DSM) (Thermo Fisher Scientific). Hemi-thoraces were then incubated in a staining solution consisting of 5 μM MitoSOX Red (Thermo Fisher Scientific) and 100 nM MitoTracker Green (Thermo Fisher Scientific) in DSM. Staining was done for 12 min at room temperature and then samples were rinsed two times for 30 s each wash with DSM. Samples were quickly mounted in DSM and imaged within 10–15 min using identical confocal microscope settings."

I'd suggest working without fixation if your set up allows it. If you have to fix your samples then make sure you work with a version of the mitotracker dye that allows fixation steps.

Good Luck,

Sebastian

Hi Sebastian.

I am trying to use mitotracker Red FM for my experiment, but i also had some penetration issues. Do you find any method to solve the problem? Do you mind to share your protocol?