Hi Carlton. I really don'y know what you mean with "general protocol". There are several specific protocols for mitochondria isolation and we had successfully used a couple of them in human brain tissue. Also we have performed mitochondria IHC and IF. If you wish you can check it at http://bit.ly/1l0FVEY better described in the supplementary material. If you find it useful and need more details let me know.

The problem is not the isolation protocol. Zebrafish is very tiny (imagine its brain) so I think you should collect "n" numbers of brain "per group" (if you are going to treat them) in order to have enought material to isolate mitochondria.

Erika is quite right, of course. Whether you isolate mitochondria from cell culture or tissue you have to depart from a relatively large amount of original homogenate if you want to have a detectable mitochondrial fraction.

Dear Carlton, my best congratulations! The task you are going to do is really tough and interesting.

Although I have never isolated the zebrafish’s brain mitochondria or any fish. Though, I have isolated mitochondria from oysters. That was an interesting experience. Anyway, to use a fish brain as a source of mitochondria will be easier but not that easy. Erica Cione has mentioned one of the problems.

I will share with you if not the experience with a fish, but rather my experience of working for 15 years with different brains and spinal cord tissues. In order to isolate PURE mitochondria you have to have a lot of fish’s heads and a microscope to extract brains. At least you have to start with several hundred milligrams of the tissue. Collect the tissue into a mixture of liquid and frozen isolation buffer and have 0.1% BSA in the buffer (Sigma brand A4503). BSA will protect from oxidative damage. In general (from observations) the fish’s brain has a lot of unsaturated fats. You have to use a Dounce homogenizer of about 25 ml volume (from the bottom to the wide part) and a loose pestle. With sharp movements up you create low pressure and help to release mitochondria from synaptic junctions. Slowly move the ball to the bottom and repeat the sharp movements about 25 times. A simple glass/Teflon homogenization might work also but may not give good yields. You have to try. I expect that the mitochondria will be very small, therefore use 800 g for the first centrifugation, save supernatant, re-suspend the sediment in half of the initial volume and spin again. Combine the supernatants and spin at 12000 g. The derived sediment, which contains a lot of fats, suspend in 2-3 ml 10% Percoll (V/V) place into the tube for the Beckmann-Coulter bucket rotor, and using a long syringe needle place underneath 6 ml of 23% Percoll. Spin for 15 min at 31000 g. The sediment of mitochondria rinse once with the isolation buffer at 16000 g to remove the remaining Percoll. Percoll might interfere with the protein assay. To prove that you have mitochondria you have to measure for some specific enzymes, most commonly cytochrome oxidase.

Good luck!

Thank you all for your kind explanation and answers, i just wonder, will the kit provided by the companies for mitochondrial isolation are good to use in such experiments? or the procedure using percoll would be better to do.

Dear Carlton,

Kits are designed for stupid and lazy people.In addition it is a waste of money because people who use kits for isolation have no control over what they do.

I agree with Alexander for both, protocol and kit opinion

Thank you professors, i understand the importance and i accept the opinion of you people. thanks a lot.

You can choose an iso-osmotic buffer having required pH for isolation of mito. from brain cells. The above information can be found at the related publications. If the study is not meant for mitochondrial respiration and you are not interested with matrix proteins, then with less caution with the above information, you can easily isolate mito. by differential centrifugation methods. For, isolation of intact mito, you have to careful about the osmotic pressure and pH your isolation buffer.

Carlton Ranjith ·: Was your attempt in isolating mitochondria from zebrafish successful?