I'm working with NT2 cells and I use mitrotracker red (400 nM) to stain the mitochondria. I incubate it with cell medium for 30 min, fix with 4% PFA, permeabilize with PBST (0.2%-0.5% Triton x-100), and block with 5% goat serum or FBS. However there is always some staining in the nucleus as well... Is it normal?
Hi Ivana,
For protocol you may refer to Cold Spring Harb Protoc 2011; doi: 10.1101/pdb.prot5648.
Regarding the staining of nuclear area, it is completely possible to observe mitochondria uptake in the nuclear area at low magnification (low NA) as you are essentially capturing signals even above and below the nucleus (visualize cell as a 3D entity). For more clarity in epifluorescence microscope you may use a higher NA or oil objectives. Further confocal microscopy can get rid of additional out of focus light in low NA images.
Ivana Zheng ,
Being agree with the previous answer, I have to add that you should also consider the fact that mitochondria actually may concentrate tight around the nucleus, for example during the mitoptose.
Biswajoy Ghosh Maria V. Kozlova thank you both for your input! I realized that mitotracker red FM, the one that I was using, doesn't work well if I fix the cells afterwards. I tried now with Mitotracker red CMXRos and it works perfectly!
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