For confocal microscopy we are trying to capture signal next day of staining. But getting very weak and nonspecific staining.
You need to provide much more info.
Which mitotracker? Are you fixing the cells? If so, how? If not, how are they kept? Are you using any pharmacological intervention? Any chance you're bleaching them?
I am using mitotracker deep red. Yes I am fixing the cells after staining by 4% PFA. No I am not using any pharmacological intervention no bleaching has been done.
I've noticed mitotracker signal get dimmer after fixation when using the CMXRos variant. I am uncertain about Deep Red FM, but I addressed the issue by increasing my final concentration from 100 nM to 250 nM.
Mitotracker signal does get weaker and unspecific after fixation. In our hands it works best on living cells which are freshly stained. Increasing the concentration helps to a certain degree, but also increases the unspecific signal. There are certain mitotrackers that are better suited for fixation, like the cytopainter from abcam.
Good luck :)
MT Deep Red is supposed to be somewhat retained after fixing. Of course, there are no guarantees and the fixing process can alter things as well.
Since presumably you already have the dye, I would try alternate fixing approaches, such as ice-cold methanol or acetone. Or even just tweak your PFA procedure.
You may be able to get something to work.
Otherwise, look into another dye, like the CellLight system, or an antibody.
MitoTracker Red has fixed well in my hands and has retains good signal.
MitoTracker Green does not fix well
MitoTracker DeepRed I have mostly used for live cell imaging.
Love those dyes!
https://vimeo.com/61159404