Dear All,
My goal is to visualize a certain protein(known to be expressed in both cytosol and mitochondrial) in order to determine its sub-localization in mitochondrial. (Electron microscope Immunogold labelling)
Before sending the isolated mitochondrial sample for immunogold staining in the EM
Electron microscope facility in our institute, they require us to show a IF staining basically to show the antibody specificity.
I haven't find right buffer to permeabilize mitochondrial and fix the cell for immunostaining.
Besides, any body know what type of method(or buffer) can make pore at outer membrane of mitochondrial at diameter of 20nm.(to let the gold nanoparticle in)
I heard that electrophoresis can make a 50nm hole on membrane. But does it practically for downstream experiments?
Thank you so much in advance.
Kai
hi,
To fix the cells usually we use 4% Paraformaldehyde for 20min, followed by 2% triton x-100 for 3min to permeabilize the cells after that use your fluorescent stain.
i hope that help you
We use the above mentionedfixing solution but for anywhere between 5-10 minutes and 0.1% triton X-100 for 10 min to permeavilize the cells followed by 3 PBS washes for ten minutes each and 30min in blocking solution before treatment with antibody.
You can use 0.3% Triton X PBS to permeabilise the membranes, but probably it could be to strong to preserve the fine structure of mitochondria, if you will use them for EM. I suggest you a more gentle method: 0.1% PBS tween, who will permeabilise the outher membrane without affecting the structure of the intracellular organelles.
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