I have spleen samples from infected mice which are currently frozen in OCT. Ultaimtely I would like to use immunohistochemistry to image spleen slices and see the ROS within different infected/uninfected macrophage subtypes. I have found a protcol that allows fixation of cells whilst maintaining the ability of DCF to stain ROS (using 10% Methanol), so the IHC itself shouldn't be an issue. My question is: are ROS likely to be stable inside macropahges within spleens frozen in OCT, or will they break down?
I was only able to evaluate ROS in unfixed OCT samples that were flash-frozen using liquid nitrogen within less than a minute post dissection. While unfixed OCT stored at -80 may produce some signals whatever findings you obtain may be criticized as unreliable. I'm also not sure how meaningful would be ROS observation in fixed material. ROS is highly reactive and may quickly disappear reacting with atmospheric oxygen. I would inject the staining the agent directly into the mouse bloodstream while it is still alive then dissect and cryosectioned the spleen.
Dear Ryan,
I completely agree with Antons comment.
ROS measurements are done routinely in our lab mainly with different ex vivo cells from the innate immune system, but it took a long time to set it right.
One of my concerns about ROS level determinations either with Microscopy or FACS is, that they are only snapshots of a rapid response. Macrophages, neutrophils and PBMCs react very quick (at least after infection and PMA) with ROS production (peak after 30 min) and end approximately after one and a half our. I would always recommend ROS measurements in a plate reader with kinetics instead of making "ROS-Snapshots". Moreover you do not encounter the problem of fixating the ROS probes.
There are ROS probes, wich I would not recommend.
The problem with luminol is, that it freely diffuses over cell membranes. So by using this substance you get a "overall ROS measurement". The same goes with the generally used DCF substance H2DCFDA, which a lot of people use. This also freely diffuses over membranes, so with that DCF probe you only detect "overall ROS" in the cell.
For both ROS probes derivates exist that are not cell permeable (isoluminol) or are retained only in the cytosol (6-Carboxy-DCF). The protocol for Isoluminol also works fine with Amplex Red (measures specifically extracellular H2O2).
Not all cells produce ROS in the same amount, in all compartments with every stimulus. What induces ROS in one cell type might not induce or even decrease ROS production in another or perhaps with other kinetics. If you know a stimulus that works in your other experimental read-outs and this stimulus does not induce ROS production with the same concentration, then there is simply no ROS production that can be detected.
For any questions, informations or technical details for ROS measurements and how these measurements look in a plate reader (in macrophages with different stimuli, scavengers and in different compartments), I would like to highly recommend our paper. 70 % of it is more or less about ROS measurements in a plate reader. The protocols described there worked in our hands with macrophages, neutrophils, human PBMCs, Microglia and MEFs.
https://stke.sciencemag.org/content/12/568/eaar5926
Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO. Science Signaling (2019)
For a overview of ROS in general I can recommend:
Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling (2012), Paul D.Ray, Bo-WenHuang, YoshiakiTsuji
ReviewROS Function in Redox Signaling and Oxidative Stress (2014), MichaelSchieber, Navdeep S.Chandel
Please feel free to ask any further questions or add your experiences.
All the best,
Marc
Excellent, thank you Anton Lennikov and Marc Herb very much for your answers-they were very helpful indeed! Ryan.
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