Hello,
I am interested in studying the damage to mitochondria in zebrafish larvae. I have been searching for antibodies for zebrafish mitochondrial proteins (Mitochondrial Marker Antibodies) to study the damage to mitochondria but found none.
Any idea or suggestions on how to study damage to the mitochondria in the zebrafish larval model?
Thank you very much in advance!
You can use fluorescent mitochondrial dyes. They should work in all species, as their mechanism is based on fundamental physico-chemical properties of mitochondria. Using TMRE or TMRM you can compare the level of mitochondrial potential between different groups/treatment. This will give you information if mitochondria respiration is compromised or not. Using MitoTracker Green you can assess the spatial organisation of mitochondria (many papers showed that fission of mitochondria is associated with deterioration of their function). You can further use fluorescent dyes for reactive oxygen species (DCF-DA, for example) to assess one of the consequences of mitochondrial dysfunction - oxidative stress. Another important function of mitochondria is regulation of calcium homeostasis. Using FURA-2 for example, you can assess the levels of calcium in the cells.
The main limitation - you need live cells for all this protocols.
Another possibility is use "Seahorse" to measure respiration of cells. Using a set of mitochondrial inhibitors you can detect the compromise of mitochondrial function in this set up.
Third possibility - electron microscopy.
Good luck)
Thank you very much for the suggestion Stanislovas S Jankauskas and hope one of the fluorescent mitochondrial dyes will work for my experiment. I use live animals (zebrafish larvae) for studying the mitochondrial function and I have to figure out how to use these dyes to stain the mitochondria.
That is right, "Seahorse" for measuring respiration of cells is a very good method and there are a couple of other methods for measuring oxygen consumption rate (OCR). I am using these for the same. I feel that and the reviewers might ask me to confirm the same with supporting evidence and hence mitochondrial markers using microscopy.
The electronic microscopy is good but it is time-consuming and availability (easily), and expertise.
If I can study mitochondrial morphology (using suggested dyes) and the mitochondrial number in control and an experimental group that should be sufficient experimental evidence.
Thanks again.
Hi Stanislovas, I have a follow up question. Do you have any papers to suggest which would describe the method of using TMRM in zebrafish? It would be greatly appreciated.
thanks
Hi Ashok Aspatwar did any of the fluorescent probes had work for you in order to assess mitochondria in zebrafish? I am at the same point you are last july. please let me know if you succed in this analysis.