Hallo,

I think you should try to separate nuclear extract from the cytoplasmic one to confirm that your protein is really localized in the cell nucleus, by western blot, using specific marker for each isolated fraction.

You can also use a nucleolus, Nucleo,Golgi and Cytoplasm marker for immunofluorescence and analyze the possible colocalization with confocal microscopy.

Hope this could help!!

without confoc, you can not say this is in nucleus (well might be the pic is confoc, i do not really decide).

Chances are that stain is inside the nucleus, since both the nuclear counterstain and the spots themselves are pretty much in focus.  Looks more like PML bodies or DNA damage sites than nucleoli to me, as nucleoli are often more variable in size.  Plus, assuming the counterstain is DAPI, if you don't saturate the images, you should see holes in the DAPI image corresponding to where the nucleoli are- do these correspond to your immunofluorescent spots when you separate the two channels?

Hello,

Agreeing to above remarks, safest way to my mind is to subcellularly fractionate and cross detect on WB with known organelle/fraction markers and confirm this with Imaging.

To cross check the fractionation results by microscopy, ideally a tagged construct should be used to exclude AB mislabeling in WB. It will be also nice to demonstrate and confirm the usage of your AB for immunohistochemistry.

Good Luck!

Hi,

the most confident methods is the transmission electron microscopy immunostaining using for instance, colloidal gold tagged secondary antibody. The resolution is better than confocal laser microscopy and you can check if your protein is in the inner or outer sheet of lysosomal membrane, mitochondria, etc, etc. If you have the transmission electron microscopy in you core facilities, ask to the lab manager how to proceed. The sample processing is quite tricky but usually is similar to the conventional immunocytochemistry, by using mild aldehyde fixation and embedding in hydrophilic resin (lowycril, LR-White, etc). Good luck.  

Hi

It does look nuclear, but I would not assume its nucleolar (I like the suggestion above). Subcellular fractionation will show where this protein is enriched, but cross-contamination is always a possibility and a  problem. Plus, if you are working with cell cultures, sample size will be an issue. Immuno-EM will settle it once and for all, but it is tricky. I would suggest going for Immuno-EM (specially if you have a facility or can get help from someone who has experience), but while you are waiting for those results might want to repeat your experiment different fixations (4% PFA vs. -20ºMetoh), permeabilization and blocking agents. We had a rather hard time with glutamilated tubulin while looking for primary cilia (see link).

Anyways, Good luck!

Article Shh Signaling through the Primary Cilium Modulates Rat Oligo...

Dear Ding-Sheng,

I agree with most comments and if you really want to resolve if your antigen is behind the two nuclear envelop membranes and understand were it is within or outside of the nucleus I can only refer to the only technique which has the "spacial" resolution - to Immuno-EM on non permeabilisation material and ideally after Cryo-Fixation (High pressure freezing followed by freeze-substitution etc.) not chemical fixed, since some antigens are redistributed during chemical fixation process.

If your protein is close to the nuclear membrane located or inside/outside lysosomes etc..you may even need to take TEM Tomograms of Immunolabled ultra thin sections (50-100nm) to be sure that the "antibody" labelling is at a specific membrane site etc.. (Comment: IgG are around 12-15nm in diameter and a double sandwich of primary and secondary antibody has a localisation radius of about 15-20nm - you can shorten this by using Fab-Au fragments to about 5-6nm). An other possibility is to use CLEM techniques to localise with the help of LM techniques (also SR-LM) the location (eg. of GFP or expressed fluorescence proteins) and finally use (it is always essential to add this too) - immuno TEM - to validate the location area (locus) and the "context" which a LM or SR-LM approach can not show.

with best regards

Roger