I am curious what is the extent of the alternations in mitochondrial network structure after cell culture fixation - is it negligible or not? What is the best protocol for fixation of cells to observe 3D mitochondria structure? I got A549 cells stained with mito DsRed2. Will the mitochondrial network structure (tubules, branching of tubules, shape of sole mitochondrial) be preserved well after fixation? I have observed interesting alterations by one focal plane live cell imaging but would like also to create high quality 3D pictures so that is why I need fixation. I will be glad for all your thoughts and experiences on this subject.
We regularly produce 2D and 3D images of HeLa, U20S, and U251 cells expressing mito-YFP or with various stains. In our lab, we have found that protocols that stress cells (even for less than a minute, such as the temperature change when taking the cells out of the incubator) can lead to mitochondrial fragmentation quite rapidly. While fragmentation is usually negligible under standard PFA fixation procedures, I prefer to fix my cells directly in the CO2 incubator by adding PFA to a final concentration of 4%.
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