Hi everyone,
I'm having some issues with an immunofluorescence staining of the mitochondria in murine cancer cells. I've tried both a MitoTracker and a Tom20 antibody and the signal is always quite diffuse.
The MitoTracker staining is very nice in live cells, but the quality gets very bad when I fix the cells. The problem is I need to actually detect the colocalization (or absence of) of a certain protein with the mitochondrial membrane, so I have to fix my cells and it just doesn't seem to work.
I thought using an antibody such as TOM20 that stains the mitochondria would work better, but the results are less good than I expected.
I followed all the steps of the IF very accurately, extended the duration of the washing steps to 5 mins each, but the signal is still not good enough, as I can't seem to resolve the single mitochondria.
I would greatly appreciate any help. Thank you!
hi,
If it suits your study context then you can explore staining such as TMRE, TMRM, Rhodamine 123 or dihydro-rhodamine.
Regards
Saurabh
Saurabh Mandal Hi, thank you for your suggestion. Do you know if those dyes are fixable? As far as I know TMRE and TMRM are not compatible with fixation as they only stain functional mitochondria.
MitoTracker actually performs very nicely in live stainings, but the fixation seems to alter the staining.
I did this. Make sure and read through the product lit. I used a red mitotracker that I think was fixable in paraformaldehyde.
They may be selling fixable Vs live product?
Mihai Valache you are right, TMRE and all, are do not need a fixation step. They are fluorescent dyes, it works directly.
It seems you are facing a problem with fixation, that particular step can be worked out by trying either different fixating agent or a serially diluted fixation method that could help.
Regards
Saurabh
Hi,
In IF, I think you can use an anti-ATP5 antibody, like this one : https://www.abcam.com/atp5a-antibody-15h4c4-mitochondrial-marker-ab14748.html.
Best,
Marie
Hi,
I used to measure mitochondrial membrane potential using MitoTracker Red CMXRose on live cells which was safe to fix with PFA (in my case 4% PFA) for confocal imaging. Make sure your MitoTracker is compatible with fixation, particularly with PFA, as once you fix a sample, it is fairly stable and shouldn’t be a problem to stain for any other marker(s).
Best of luck,
Kathy
Dear Mihai Valache
Live imaging is the best but incase if you do need to fix then Methanol or PFA might work. I am not sure which fixative you have tried so far.
In our experience, the effect of fixative on the mitotracker stain also depends on the cell type being used. So better to test both fixatives for your target cells.
there have been previous discussion threads on researchgate which might be helpful for you. pls have a look for more details-
https://www.researchgate.net/post/Can-MitoTracker-CMXRos-be-fixed-using-Methanol
https://www.researchgate.net/post/What-is-the-best-fixation-for-fluorescence-microscopy-of-mitotracker-in-HEK-cells
good luck !