Hi Timmy,

Some of the cells appear to have high background. The staining should appear more like a halo around the nucleus. If you look very carefully at the cells that appear as "filled oval shape", you will notice that the lamina is actually staining more brightly than the interior of the nucleus. I am not sure why there is so much background in some, this protocol appears to look the best:

"Fix with formaldehyde prior to staining. Cells were probed without (control) or with an antibody recognizing Lamin A/C (Product # MA3-1000) at a dilution of 1:200 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody"

in some instances, they report using the primary antibody at a much lower concentration.

Kind regards, Lora

I agree with Lora Benoit . In addition, to me it looks like the "ring" pictures are taken with a confocal microscope, which makes "slices" as images, and thus there would be much less background in the nucleus. The "bright" pictures might have been taken by a non-confocal fluorescent microscope, means it takes the whole thing, creating more fluorescence in general and thus making an illusion that "nucleus is stained".

Lora Benoit Yulia Panina Thank you very much for your reply!

I once speculated that the fluorescent signal located at the nuclear interior is because of the presence of nucleoplasmic lamin A/C. But seems it is not the case, at least in the following case. As I found the Lamin A/C immunofluorescences of LAKI mice, which produces permanently farnesylated progerin that leads to reduced ability to migrate to the nucleoplasm, also have the filled version rather than the ring version (attached photo from https://www.cell.com/fulltext/S0092-8674(16)31664-6 ).

I also have thought about the effect of using different types of microscopes. The attached photo was taken using a laser scanning microscope (Zeiss LSM 780 laser-scanning microscope (Carl Zeiss Jena)), will it be the reason why all the Lamin A/C IF figures in the above article end up with "filled nuclei"? Imagine if I am going to use a typical confocal microscope, but not the scanning one, to get the cross-session IF image of the same batch of cells, I should assume to see the hollow ring shape right? What do you think?

Hi Timmy, you have obviously thought this through very carefully. Confocal should give you the desired ring-like appearance. Also some of the commercial antibodies recognize a precursor form of laminitis a which can be found inside the nucleus rather than the periphery. You should check the specifics of the clone you are using to rule out that possibility if you are gunning for the ring like appearance.