I have used MitoSox in C. elegans and it works nicely, but I am unsure of if it would work in the live mammal animal. You can also use MitoSox after fixation.

Dear Brian!

You also look at

https://www.researchgate.net/post/How_to_identify_oxidative_stress_in_a_formalin_fixed_tissue_sample

Pavle Randjelovic added an answer

June 24, 2014

You can use immunohistochemistry for this. There are various antibodies for detection of markers for oxidative stress end products of lipids, proteins and DNA. 4-HNE, nitrotyrosine, 8-OxoG and so on.

Brian,

I would not recommend to use MitoSox with fixed tissue!

1. MitoSox is a membrane permeable weak acid. In live cells it distributes into the mitochondria because , as long as the mitos maintain their membrane potential, the mitochondrial matrix is the most alkaline place in the cell. Once MitoSox arrives in the matrix it gives off a proton. Now electrically charged it is no longer membrane permeable which effectively traps it inside the mitos. Thus, for proper localization it depends on the presence of membrane potentials which are missing in fixed tissue.

2. MitoSox becomes fluorescent after reaction with superoxide. The stain is meant to report superoxide as it is produced by the mitochondria, typically by activity of the electron transport chain (normal or pathologic). Fixed mitochondria are not metabolically active any more and therefore should not be able to produce superoxide any longer. Superoxide is diffusible, reactive (ROS - duh!) and therefore has a very short half-life. Thus, the superoxide that has been present at time of fixation will be long gone at the time you stain your fixed tissue slices.

Anything you find with MitoSox in fixed tissue will probably be caused by non-specific effects, such as autoxidation, and will be more confusing than useful.

I recommend to follow Alexandr's hints and concentrate on immunodetection of permanent ROS-damage to the tissue (protein, nucleic acids).

Good luck with your project!

Bernhard

Ernst-Bernhard Kayser ,

What I meant to write you was: Thank you so much for this detailed explanation as to how the MitoSox stain works and why it wouldn't be feasible to perform it in FFPE tissue. This really helps me understand why it is only applicable to live cells as well as gives me easily digestible semi-mechanistic information to consider in the future when working with any special dyes. Great answer and tip!