I recently prepared some fixed cell samples for imaging. I need to observe the mitochondrial morphology. I overexpressed a GFP tagged protein, which localizes in the matrix of mitochondria. As is shown in the figure (only show GFP signal). When I did observation with live cell, the mitochondria looked tubular and healthy. However, after fixation, the mitochondria became fragmented and fat. Some of them like donuts. What is wrong with the fixed sample?
Here is the fixation process:
1) Wash cell 3 times with PBS.
2) Incubation with 4% PFA for 15 min at room temperature.
3) Wash 3 times with PBS.
4) Incubation with 3% BSA containing 0.2-0.5% Triton for 30 min at room temperature.
5) Wash 3 times with PBS.
6) Incubation with primary AB overnight at 4 °C.
7) Wash 3 times with PBS.
8) Incubation with secondary AB for 1 h at room temperature.
9) Mount.
Hi Dongxue,
Fragmentation of mitochondrial network is know reaction to stress.
Article Mitochondrial fragmentation and network architecture in dege...
Try to find ideas how to prevent it here
Article Methods for imaging mammalian mitochondrial morphology: A pr...
Daniil
I wouldn't go over 15 minutes for fixation. What are you using to mount the sample? We have good luck with Immu-mount from ThermoFisher. If you pre-warm your PFA, you actually don't need to increase your fixation time, as the formalin will work faster at a higher temperature. When your samples are fixed and prior to adding the antibodies, I would check the cells under the microscope to see what their morphology is. Maybe your staining prep is the issue, however we follow the same guidelines for our staining.
Hi Xuxia,
Xuxia Wu
I find the permeabilization step is very important. Now I use 0.5% triton to permeabilized the cell for 30min immediately after fixation. I can see very normal mitochondrial.
Good luck to you.
Hi Dongxue,
Dongxue Hu
Thanks for your advice.
In fact, in my test, the mitochondria have become fragmented immediately after the cells were washed with PBS for the first time. Interestingly, the mitochondria have gradually restored the tubular network after being placed in the PBS for a few minutes. However, the morphology of mitochondria is still different from the washing before. It seems that washing with PBS is no a good choice ?
Hi Xuxia Wu
In my hand, washing with PBS does not change mito morphology. I guess it is not the problem of PBS, if your PBS is correct.
Is your green color mitotracker dye? I use stable cell line that express mito-GFP or RFP.
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