I have tried mitotracker staining of leishmania. It is giving very nice staining when parasites are live. When I fixed leishmania with 4% Paraformaldehyde, it looses its mitotracker staining. Also I have tried cytochrome staining in live leishmania, hardly 5% of leishmania are taking staining. I want to stain both mitotracker and IHC of Cytochrome 'c' simultaneously. Kindly suggest me some protocol.
Are you sure that the MitoTracker that you are using is fixable? Most are fixable but some are not - see page 3 of the MitoTracker manual (available here http://www2.cbm.uam.es/confocal/pdf%20plasmidos/mitotrackers.pdf ). In my experience MitoTracker Red CMXROS works well (i.e., fixes) in T. brucei, but MitoTracker Green FM does not. Hope that helps.
Hi,
I am using MitoTracker® Red CMXRos for staining mitochondria of leishmania. When cells are alive it works well but fixation causes blurred staining of mitochondria.
MitoTracker Red CMXRos does not come out of the mitochondrion once it is in there (it covalently binds to protein thiols in active mitochondria) so if you are seeing blurred staining only after fixation it might be that your cells are dying and degrading before they are fully fixed.
Hi,
I will try to fix leishmania with different percentages of Formalin and paraformaldehyde after mitotracker staining. I will let you know as I get my proper Cyt C and mitotracker staining.
Hi Durgesh,
have you some new findings? I've go the same issue..
Thanks,
Tina
Hi Tina,
I haven't got proper mitotracker staining in fixed cell. You can do one thing, keep leishmania at +4 degree celcius for some time. Immediately transfer them on slide and take images. Lower temperature reduces mobility of parasite for some time. This will solve your problem of imaging. Quenching problem can be solve by DABCO treatment.
Hello,
I don't use lesihmania but I work with SH-SY5Y cell line. I've try to stain CYP450 and mitotracker at the same time. First I used mitotracker CMXRos with live cells, then I fixed with 4% PFA and made the IHC for CYP450.
I did it first in one lab and it worked perfectly, you can clearly see the mitochondrias very well stained. Then I came back from my stage in that lab and tryed to do the same. I used the same cells, same protocols, and same products. The only thing I have change are the coverslips and the microscope. Now I have the same problem than you, I can see some of my cells well stained, but my samples have a strong red background that doesn't allow me to take a nice picture.
Any suggestions?
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