In my lab we work with multiple vascular beds using pressure myography to test endothelial dependent vasodilation (i.e. acetylcholine and/or insulin). It is of our interest to detect NO levels after stimulation using confocal microscopes and we have used protocols used in papers and also made changes from answers to questions here in RG. So far we have not been successful, I would really appreciate if someone could give me an advice or a protocol that works.
In short what I want to do is:
1. After temperature equilibration, pressurized arteries under static conditions at 37°C will be challenged with 80mM KCl to test vessel viability.
2. Arteries are washed 3x with PSS and wait for 5 mins.
3. Myograph chamber is translated to confocal microscope with temperature controlled system set at 37°C and we wait for 1 mins to the vessels to adjust to the new environment.
Here is my questions:
Some protocols say:
Incubate in acetylcholine (for exmaple) for 10 mins and after that incubate with DAF-2DA (5µM) for 30 mins and both steps at 37°C. Wash 3x for 1 min and fix with 4% paraformaldehyde and then add DAPI and image. You will only have a time measurement of NO.
A different protocol says to incubate with DAF-2DA (5µM) first and then take an image at t=0, add acetylcholine and incubate for 10 mins and take an image every 30 mins after that.
Those protocols are either for cells or aortic rings using en face mounting to image. What I want is a pressurized vessel, so I think the best option is to add DAF-2DA intraluminally and add acetylcholine to the vascular bath. But what option is the best, Should I incubate with acetylcholine first, or with DAF-2DA first? Should I fix my artery before imaging or should I do a time-dependent experiment? Is there any normalization taht I should use to help with the analysis? Is there anything else I should pay attention?
Thank you for you time in advance.
Francisco I. Ramirez-Perez
Hello Francisco,
If you are planning on monitoring NO generation real-time, then any grossly invasive procedures seem not very productive for your goals.
Given that NO is actually a diffusive gas, prolonged and elaborate procedures prior to detection add to possibilities of artificial outcomes. Therefore, the additional staining like DAPI is actually not advised, if NO staining itself is already challenging enough. It also concerns the magnification of info at which you want to show -- NO as detected by DAF-FM, for example, is best presented at the whole cell or cell population level (tissue sections), not at the subcellular level (thus, counter-staining with a nuclear DNA dye does not help much).
Probe incubation followed by prolonged PFA fixation and detection is not ideal, because these procedures tend disrupt cell membranes and alter permeability signficantly. DAF series probes are small molecules, and fixing will reduce the chance of their fluorescent products being detected (false negatives).
Please consult our paper on staining mouse vessels with a fluorescent probe for peroxynitrite (ONOO-), an RNS derived from NO, using a protocol involving live tissue probe staining by perfusion, brief PFA fixing, embedding, cryosectioning, and finally confocal imaging. (See details in SI). This method should work for other small-molecule fluorescent probes provided they are sufficiently retained and survive the fixation and wash steps performed by the experimenters.
Article Molecular Imaging of Peroxynitrite with HKGreen-4 in Live Ce...
pubs.acs.org/doi/suppl/10.1021/ja504624q/suppl_file/ja504624q_si_001.pdf
In general, if you are interested in test acute drug effects, the procedure would be:
- pre-loading the NO probe (to a final recommended conc.) into the cells or isolated/bathed tissue. Typically 30 min will do. Wash is usually not needed at this stage unless the commercial protocols state so, or you can test it out in imaging, to see how much fluorescence survive following washes.
- add the drug to simulate NO production. Allow sufficient time for build-up.
- terminate the incubation by a lavish wash (2x original volume for example) or directly proceeding to PFA fixation (5 min, on ice; 4% PFA in PBS).
- Wash lavish 1-2 times to remove residual PFA (when dried, or concentrated, this becomes autofluorescence or other artifacts).
- proceed to OCT embedding and normal cryostat sectioning (low temp.). Do not use paraffin embedding, which will invariably fail.
- mount sections onto a glass slide with coverslip (plus anti-fade Gold, Invitrogen), for imaging (confocal, magnification at 40x or 63x).
Good luck.
Thank you Nai-Kei for taking your time to help me with my question. I'll try what you recommended me to do and hopefully it will help me with my experiments. NO is really important in my research and we have issues using several detection systems, it is hard to get reliable results so far, once again thank you for your help and hopefully this will help.
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