I tried staining 3D cancer spheroids using F-Actin stain but the F-Actin filament were not well distinguished as lines. It looked like mitochondrial or lysosomal stain. Any ideas why it turned out like this? how can I solve it?
There are three things you can consider. First: make sure the fixative is fully rinsed out;it quenches the fluorescence.
Second: is the spheroid fully permeabilized? Try increasing the permeabilization (detergent) time. Them fully rinse out the Triton detergent.
Third: if you cannot use confocal microscopy, slightly compress the sample with the cover slip and seal it (clear nail polish is fine) to hold it down. This will give less curvature in the plane of focus.
Ellen A G Chernoff I usually do permeabilize with 0.2% triton-x for and block with 1% FBS in a single step for 20 minutes at room temperature. So I will increase the permeabilization time. But would the temperature also have an effect here?
If you use a labeled phalloidin probe, it binds with very high affinity. You can leave out the FBS, no blocking is needed. I do not know for certain that it interferes, but I have never used it, even on large spheroids. I have always permeabilized at room temperature, as you do. Increasing the temperature, perhaps to 37C would speed up the process. So would increasing the Triton concentration.
Mine is a cellmask actin tracking stain. but I don't think there is a big difference between the protocols for the two stains, right?
I have one more question, please. Do you think poor washing after staining can lead to poor quality images like the one I shared in the post?
If you only want to see F-actin, you should use phalloidin conjugated to TRITC or Texas Red. Fixation in methanol (4 degrees C) followed by acetone extraction (-10 degrees C) for 5 min or so, should solve your problem, both with staining and background.
Ayman A. A. Yes, poor rinsing of ixative and permeabilizing agents causes these problems. I see it frequently in the lab course I teach. (We use Rhodamine- phalloidin because it’s relatively cheap, though it requires fixation and permeabilization).
You can get phalloidin from Invitrogen or SigmaAldrich. It's less than an AB.
Be careful with which fixative you use if you want to stain with phalloidin - methanol and ethanol will destroy phalloidin binding.
"To create the correct fixation conditions for phalloidin binding paraformaldehyde must be used as the fixative because it retains the quanternary protein structure which is necessary for high affinity. Methanol destroys the native conformation and hence is not suitable for actin staining with phalloidin."
https://www.cytoskeleton.com/actin-staining-techniques
But I guess you have some binding unless the signal you show in the image is just autofluorescence. Is it possible that the cells just don't have strong distinct actin filaments when in spheroids? The cytoskeletons of the spheroids I have imaged look nothing like what you see when the cells are grown on glass. More "tangled" in a way...
I have used methanol/acetone fix/extraction to show F-actin structures for a very long time. Never had a problem demonstrating them within the cytoplasm.
Hi Sheldon R Gordon , please would you be willing to share your protocol for methanol fixation for phalloidin staining? I see in your papers descriptions of paraformaldehyde fixation then acetone extraction, e.g.
https://link.springer.com/article/10.1007%2Fs00441-020-03229-2#Sec2
Article Microfilament Disruption in a Noncycling Organized Tissue, t...
but I don't have access to all your articles to find the methanol fixation version. It would be great to be able to use phalloidin on some of our ethanol-fixed samples. Thanks for your help!
Ayman A. A. Another aspect you might want to consider is your imaging set-up. Spheroids give lots of scattering. Are you using a widefield or confocal microscope? What scale is the image you showed? Are there any other fluorophores in your sample that do show good spatial resolution? What is the spheroid mounted in? Consider trying to match the refractive index of the mounting medium better to the sample. Glycerol works well for me with spheroids, but we have a glycerol immersion lens on our confocal microscope, so if you just have water or oil immersion you will need to adapt to those.
I see from the CellMask actin tracking product website that is suitable for live imaging
https://www.thermofisher.com/order/catalog/product/A57249#/A57249
so I would not expect permeabilisation time with Triton X-100 to be a problem. The CellMask datasheet has an image of a stained spheroid which doesn't show any filamentous actin either!
Don't use methanol when you are using phalloidin. And yes, it should stain F-actin beautifully.
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