The spheroids are made of MDA-MB-231 breast cancer cells which are themselves producing GFP-fusion proteins. Only very portions of the periphery can be seen by the confocal microscope, whereas the huge middle part (where the actual ball of cells is) don't show up anything. I have checked under a normal fluorescent microscope and I do see a green solid sphere of cells.
The spheroids are about 400-500um in diameter. Could it be that the spheroids are too packed or dense for the confocal microscope to "slice through"?
p.s. I anticipated that there will be a small hypoxic region in the middle, and have discounted that as a major contributor to the problem.
I agree with the above response. I use a standard inverted fluorescence microscope with a motorised Z-plane to take a crude Z-stack. Presumably the more intense light source allows fairly clear imaging within the spheres. I grow spheres up to 1mm and see signal in these, even using destabilised GFP reporter cell lines.
Hello Dear,
I observed a similar type of staining with my spheroids and I thought that may be antibody was not able to penetrate fully. But in your case since cells are endogenously expressing GFP the main reason is due to incomplete light penetration as suggested by Henk. I opt for simple fluorescence microscope, used TBS for washing and antibody incubations and was able to capture nice images in inverted fluorescent microscope. I didn't go for confocal after that. And yes, cryosectioning is a better option but a labour intensive procedure.
Hope it helps !
Your spheroids are really big to be transparent. You may try to cut them in half and see (with a confocal) if there is any fluorescence coming from their centers. Or you may try to fix, permeabilize and 'clear' them with some established protocol (CUBIC, SCA\E, Clarity, iDisco, etc, etc - basically lipid removal and index matching to reduce scattering) to render them transparent, though that may be difficult and time consuming...
Good luck!
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