For isolation of mitochondria from rat hippocampus, chilled isolation medium (0.25 M sucrose containing 10 mM Tris–HCL, pH 7.4, 1 mM EDTA, and BSA 250 lg/mL) is made.
Can we make this solution and store it for long? If yes, kindly confirm how long, at what temperature would we can store and use this already made chilled isolation medium ?
or it should always be freshly made ?
AND
If we place our rat hippocampus in this solution, how long can we keep the tissue in it until mitochondrial isolation from the tissue ??
Can we preserve our isolated hippocampus tissue in it for several weeks ? If no, suggest the alternative.
After perfusion with 4 % paraformaldehyde or 10% formalin solution. I keep the brain in same solution for one day and then shift it to 15%, 25% and 30% of sucrose. For crysection i take
- brain from sucrose solution and cut the brain region rostral/caudal to region of interest (ROI) with a blade.
- I put OCT compound on stage and place brain on it. and then apply OCT on all side of brain and then spray liquid nitrogen. Cover the brain for 10 to 15 min to get the desired temperature. I keep the stage temperature between -18 to -27 then
Important Note: Cutting Temperature should be 1-2ºCbelow when slide start rolling/fell down.
I have stored it in the 4 degree for a while. Just always check it for any bacterial growth before using it.
I do Para for one day and then directly into 30% sucrose for three days then into OCT/paraffin with mouse brains, but with rat brains, I did do a gradient of sucrose and then into OCT or paraffin .
You can keep the mito isolation buffer in 4degC no problem. I don't know exactly how long you can store it but I think at least 1 month should be okay. However, the BSA should be added fresh, same day and then double check the pH after adding the BSA. If possible, BSA that is "fraction V, fatty acid free" is best. If you plan to do any proteomics, consider that there is BSA protein in these isolated mitochondria samples. BSA helps protect the mitochondrial fraction from oxidative damage during the isolation procedure.
Once you place the fresh hippocampus in the solution (on ice), you continue quickly with the homogenization, centrifugation, density gradient isolation procedures which may take awhile.
The best method is given by Dr. Ronald Irwin that safeguard the integrity of mitochondria.
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