Hello,

You cannot really perform ROS detection on fixed cells. You can only perform ROS detection before fixation. It has been discussed a couple of times. ROS should always be detected in living cells and living tissues in a good state. The rationale of that has been discussed. https://www.researchgate.net/post/Can_cells_be_fixed_before_ROS_tests

Try not to use DCF and derivatives. They are advised against by reviews of the Society of Free Radical Biology & Medicine, due to many inherent problems, including chemo-instability, auto-oxidation (you get a huge fluorescence turn-on without ROS, when the "probe" is exposed to air [oxygen] long enough, to metals, strong light, etc.).

Commercial probes include hydroethedium (HE, ThermoFisher) for superoxide (the primary ROS), and some H2O2 chemical probes.

https://www.tocris.com/products/peroxy-orange-1_4944

What you can do, is that, at end point of your experiments, prepare a small bath of buffer with the probe (typically, probes are used in the conc. range of 5-10 uM). Immerse your samples in the bathe, for 30-40 min (should be enough), for detection (live cells, or tissues). Then, quickly fix your tissues/cells in PCL with PFA (4%, 4C, 5 min), followed by 2-3x of washes (PBS). Then, embed these in in OCT, either for cryosectioning or direct imaging if you have a suitable imaging platform.

A similar method was described here.

Article Molecular Imaging of Peroxynitrite with HKGreen-4 in Live Ce...

See SI:

https://pubs.acs.org/doi/suppl/10.1021/ja504624q/suppl_file/ja504624q_si_001.pdf

tAfter staining with the probes (30-40 min incubation mentioned above), you can indeed proceed directly to imaging if your samples can be easily mounted for imaging directly. Make sure the imaging session is not too long, as your cells/tissues may degrade over time (That's why fixing was advised AFTER ROS detection, not BEFORE it).

Good luck!

Hi

I believe it is impossible - PHA reacts with many ROS and what you could measure would be the remaining postoxidized molecules. For simplicity, you may try to measure ROS by DCF +/- PHA +/-H2O2 (or other oxidants as I am not sure which ROS you are trying to monitor) in a cell-free system, e.g. quarz cuvette.

Do a little kinetic and watch the spectrum changes.

meant PFA