Lysosomes / Mitochondria - telling the difference - using lysotracker / mitotracker (Sep/17/2006 )
I'm trying to identify localization within the cell and I am not sure if my peptides are ending up in the lysosomes or the mitochondria. Currently, I'm using lysotracker and mitotracker from molecular probes. Problem is... the staining is pretty similar so I can't really tell one from the other, especially under a fluorescence microscope. It isn't even that clear under a confocal. Anyone else has a problem like that before?
Any suggestions on what I can do?
Here's an image I found using both lysotracker and mitotracker. Seems to be an ultra high resolution image. But otherwise, it actually looks pretty similar (in terms of localization).
Lysotracker / Mitotracker
How abt using a mitochondrial specific antibody along with mitotracker and compare the staining.
I've actually used cytochrome c antibody and it localizes somewhat similar to the mitotracker. Let me try describing the problem i have.
imagine a venn diagram, A, B, C where B = AxC (A intersect C)
mitotracker labels A
lysotracker labels C
there's a pretty big overlap where both mitotracker and lysotracker label
- that's where my peptides are going.
- now question is: where ARE they going?
technically i can say they go to the mitochondria, but i can also say they are going to the lysosomes. but either way, i'm not convinced.
Also, i've tested different concentrations of mitotracker and lysotracker so that I use the minimum necessary to label (i.e. so I don't overstain the cells)
Has anyone tried using both before? It seems that most people use either mitotracker or lysotracker only and not both. but if both colocalizes to some extent, it would be wrong to draw a conclusion from a single staining wouldn't it?
I'm not sure if I am making sense here. Maybe I'm thinking too much unnecessarily. thanks for reading.
There is no harm in trying out both mitotracker and lysotracker assuming they can b visualised using dif. filters.
Also if u have the possibility to do EM-electron microscopy, it might b the best thing if u r unsure abt the staining.
If u could stain live cells and image it live, one could observe mitochondrial movement and mitochondrial fission and fussion. Just an idea.