I'm putting together a plan to try immunofluorescence staining of a pure isolated mitochondria sample as a control experiment. I have already successfully done immunocytochemistry with these antibodies, but I have a few questions for this next (immunomitochemistry? lol) experiment I am wanting to try:

1) What method of fixation would be best?

2) What sort of media should I suspend them in to keep them stable? Would something like MiR05 be suitable, or am I way overthinking this?

3) Am I correct in thinking that Tween-20 would be less harsh than Triton X-100 for permeabilizing the mitochondrial membranes?

4) Any big things I might be overlooking?

Thanks in advance!