Hi,

Most probably the signals which you see apparently in the nucleus is the layer of mitochondria above it. Confocal imaging will definitely help you, moreover there you can create 3D image, which will give you a clearer perception, whereby, in one of the planes you can observe the nucleus absolutely neutral with mitochondria surrounding it.

All the best!

Thanks, Suraiya.

All the best

Stefano

Hi,

I agree with your and Suraiya's assumption that the signals are most likely mitochondria in a different focal plane. Note the blurry outline of the structures opposed to the well-defined rod-like appearance in the cytoplasm, resulting from those objects being out of optical focus. If you want to be sure, confocal microscopy, possibly with 3D-reconstruction as already suggsted should give you a clear impression on the objects' z-positions.

Thanks, guys. I'll try a z-stack by confocal microscopy and let you know.

Stefano

Hi everyone, confocal microscopy did not confirm that nuclear signal was coming from above/below the nuclei. Rather, 3D reconstruction gave clear evidence that Mitotracker nuclear staining was associated with optical sections within the nuclei. Some representative micrographs will follow tomorrow. Any help/idea to explain such binding event?

Thanks

Hi again,

interesting, it appears that the stain is less specific than claimed my the manufacturer. The classical MitoTracker (red) is known to covalently bind several proteins, causing unspecific staining in a variety of organelles, see Article "Stainomics": Identification of mitotracker labeled proteins...

I would imagine the same is true for its derivatives (orange), as usually only insignificant modifications to the fluorochrome are introduced to yield spectral shifts of the dye.

On a second look, the structures' numbers, size and morphology in the Mitrotracker channel remind me of nucleoli, however those should be labelled a bit less intensely in the DAPI channel (darker "blobs").

If you need a perfectly specific signal, DASPEI may be worth a look for an alternative mitochondrial staining.

Hi Patrick. Yes, I've already noted that the structures' size and morphology in the Mitrotracker channel looked like nucleoli, however I doubt they are for the same reason You mentioned: DAPI channel should look different. Today my confocal microscopy photographs will be available and uploaded, so You can have a clear idea of the staining pattern.

Thanks again.

Stefano

Confocal microscopy images of HUVECs labelled after 40 minutes - 500 nM MitoTracker.

As clearly shown in the pics, nuclear red signal (Mitotracker) comes from within the nuclei (please, look at separate red and blue channels of the TIF files). Of course, I'll forward these pictures to Thermo Fisher Scientific technical support.

Any idea from You, guys?

Thanks again.

Stefano

Dear Stefano,

Recently I had similar results with TMRM. The dye specific for mitochondria. But in case of frog erythrocytes it was only nuclei staining.

My collegues said that it is results of TMRM charge (positive). Check a charge of mitotracker used. May be it is a reason of penetration mitotracker into nuclei.

Dear Nikolay,

as reported by the datasheet (https://www.thermofisher.com/order/catalog/product/M7510), the fluorochrome is cationic, thereby my situation may be similar to that experienced by You. I performed similar experiments with much lower/shorter concentration/incubation time and obtained images with faint signals (even though nuclei were still positive). Therefore, it seems I can't get rid of the nuclear staining so easily.

I'll update this thread as soon as I get an answer from the supplier's technical support.

Thanks for Your contribution.

Stefano

I've worked with this dye extensively here at Molecular Probes and never before have seen this staining pattern. It appears nucleolar in your images.

What is "RSV+HG" (from your image name). Is that some sort of special treatment which might be changing the dye localization? If so, have you tried a control without that treatment? Or could the treatment itself lead to such staining?

Also, I'm wondering how old your MitoTracker stock is.

Hi Jason. The Ph.D. student treated the cells with different compounds, however we found the same staining pattern in the vehicle-treated cells, so no treatment-dependent effect on the localization of MitoTracker was evident.

With regard to the "age" of the dye, I don't think that's the problem: just few months (correctly stored).

Any other possible explanation from anybody else?

PS

Still waiting for reply from technical support.

Stefano

Thanks for the additional information. Actually, I'm with Molecular Probes Tech Support, for North America (you're probably consulting with my colleagues there in Europe). The first thing I'd recommend is reducing the concentration. When I've used the dye, I found that 50nM was sufficient, for 20 minutes at 37C, in media or physiological buffer. With too high concentration or too long label time, I've seen nonspecific labeling of different sorts.

With 100 nM - 20 min, I got faint signal, but nuclear staining was still present. When you say "too high concentration" you mean also 100 nM? I've found several protocols suggesting 200 nM...

Hi Jason,

here are some images I got for control HUVECs that were incubated with 100 nM MitoTracker® Orange CMTMRos for 20 minutes. Faint signal, but nuclear labelling is still present. I really doubt I'd get rid of the unspecific signal with lower MitoTracker concentrations. Rather, I think I'd loose further the specific signal.

Waiting for a kind help.

Thanks in advance

Stefano

Yes, I think you are probably correct that if you are already pushing the lower limit of mitochondrial detection with 100nM concentration, that you probably don't want to go lower. And you're right in that plenty of researchers have used the dye in the range you have used. I tracked down your Tech Support connection. I am suggesting that they replace your unit of MitoTracker, just in case it's something to do with the dye, but I must confess that I don't know why the nucleolar labeling is happening or how to stop it, as we haven't seen it before.

Thanks so much, Jason. Really thanks.

Best regards

Stefano

Hi Jason. I've just received a reply from tech support.

"Jason says is that it is not something we have seen happening before. If the customer rules out the possibility of visualizing other focal planes by confocal , it most likely is staining nucleolus. Yet we are not sure why or how. The nucleolus could have a membrane potential that would attract the dye even through nuclear membrane or it could be the dye-charge issue. We agree it is an unexpected result. The recommendations are to lower the MT concentrations even further, to about 50nM and see if that helps."

You agreed that the idea to lower the Mitotracker concentration was not good. In fact, in Your last message You wrote that You would suggest that they replace the unit of MitoTracker.

Jason, could You please re-suggest them to do what You wrote in Your last message?

Thanks so much again and have a nice weekend

Stefano

Ah, the message didn't get through to the Tech person, Alessandra. I'll pass it along again. Would you like a replacement with the same product (I'm assuming), or a different MitoTracker?

Alessandra agreed to replace the unit (same product, different lot number from USA). You've been of really great help.

I am truly grateful, Jason. Thanks a lot.

I'll keep you updated.

Best regards

Stefano

Hi Jason. First of all, thanks a lot for Your very precious help and assistance. I finally got the new lot of Mitotracker and repeated my last experiments. As You may see in the attached figures (epifluorescence microscopy), the new Mitotracker seems to stain well at lower concentrations (25 nM, for 30 min), with respect to the old lot. Unfortunately, the nuclear signal can be still seen very clearly. I looked for others with similar issues and found the work from Duran-Prado et al. (2014): Article Coenzyme Q10 Protects Human Endothelial Cells from β-Amyloid...

The images shown in the paper seem to confirm that HUVECs may exhibit nuclear staining with Mitotracker (even though the Authors used Mitotracker Deep Red).

Others have found nuclear staining in SiHa and BMSC cells with both MitoTracker Red FM and MitoTracker Green FM (Zhang et al. Anal. Chem. 2015, 87, 12088−12095).

I am starting to think that I am not the only one seeing a nuclear signal!

Would You, please, help me in searching for possible explanations of such staining pattern? It might be difficult to get my co-localization results published with such unspecific signals shown.

Thanks a lot in advance,

Best regards

Stefano

Interesting, Stefano, and I'm sorry to see it. I have heard scattered reports through the years, as well, but I don't know why it happens in those instances and not in most.

I'm wondering what buffer or media you are using for the label step, and whether you've tried others, or if that changes the label pattern at all. For instance, what effect would slight differences in pH, salts, or serum proteins have?

I've enlisted the help of one of our long-time chemists here at Molecular Probes, and will get back to you with what he says.

Just to throw an extra monkey wrench into the conversation here...

In grad school we developed a "mitochondrial" sensor that was using triphenylphosphonium to localize but it also showed very clear nucleolar staining. I confirmed it was nucleolar by confocal co-localization with nucleolin-GFP and fibrillarin-GFP. This is all unpublished (and probably never will be), but we originally thought it might be that the moiety our sensor was developed for was also in the nucleus.

I think there must be some charge distribution specific to the nucleolus. It would make sense, considering how much nascent RNA Is synthesized there that there would need to be a lot of cation for stabilization - perhaps there is a unique charge distribution there.

Here is another example of published work with TPP (and mitotracker green) that went to the nucleolus (figure 2): http://www.mdpi.com/1424-8247/10/4/91

Dear Jason, I'm preparing a list of the complete procedure we are following to process samples for IF. I'll post here, so You can try figure out what is going on.

Jordan's suggestion is very interesting. I didn't think of that! Maybe he is right. Perhaps what we are facing here is something linked to nascent RNA-related electric charge. This is something I could discuss when showing nucleolar staining in my paper. The photographs in the literature he mentioned show intranuclear signal very clearly. Thanks so much, Jordan.

Stefano

Followed protocol:

HUVECs seeded onto coverglasses;

..................... time......................

Medium removed;

MitoTracker working solution added (concentration range: 25-100 nM);

Samples incubated at 37 °C for 30 min in the dark;

MitoTracker working solution removed;

Two brief washes with PBS 1X;

Cells fixed with PFA 4% w/v in PBS 1X at RT for 10 min;

One wash with PBS 1X;

Cell membranes permeabilized with PBS 1X + Triton X-100 0,05% v/v (PBS-T) at 4 °C for 10 minuti;

Two washes with PBS 1X at RT (5 min each);

Blocked unspecific binding sites with PBS-T + BSA 3% w/v at RT for 30 min;

One brief wash with PBS 1X at RT;

Samples incubated with primary antibodies overnight in humidified chamber at 4 °C;

Five washes with PBS 1X at RT (5 min each);

Samples incubated with secondary antibodies at 4 °C for 1 hour in a dark humidified chamber;

Five washes with PBS 1X at RT (5 min each);

Coverglasses mounted onto microscope slides, in presence of 2,5µL Vectashield+DAPI (Vector Laboratories, cat. H-1200) in the dark;

Coverglasses sealed with nail polish;

Samples observed by epifluorescence (Zeiss, Axio Imager A2; camera Leica, DFC320), and photographs acquired by b/w camera (40X) (aperture 2; Exposure: 690 ms).

Protocol seems fine to my eye, though 4C secondary I would do RT and my washes always have detergent. Mito staining looks okay there in my opinion.

Have you looked at your cells live?

No. That's a very good idea! It would clarify whether fixation/permeabilization causes the issue. I'll keep You informed.

Thanks a lot, Jordan!

Stefano

The protocol seems reasonable, and seems similar to what I've done in the past. I agree with Jordan's suggestion of looking at them live, to determine if the later steps may contribute to the issue. Overnight in primary may not be necessary for cells in culture (unless that is something you've determined to be optimal for that primary already), and I wonder if the long incubation period may lend to a relocation of the MitoTracker.

Another thought: you are sealing with nail polish in a non-curing (Vectashield) mountant. I have found that the solvent in nail polishes can have unwanted side effects on many dyes (such as completely killing GFP signal), though I haven't looked at the effect on MitoTracker. You can try with vs. without nail polish. But I would suggest changing to a curing mountant (such as to our ProLong Gold with DAPI) since it will harden for better archiveability and doesn't require secondary sealing.

Thanks for all the valuable suggestions, guys. I'll change the sealing process and the pre-fix observation, and I'll post the news here.

Enjoy Your weekend!

My best regards

Stefano

Hi, I used MitoTracker® Red CMXRos recently in HEK293FT and Hela cells, and found myself in a very simailar situation.

I've done some pre-experiments today, and found an interesting phenomenon that might be helpful. That is: I can only see this postive nuclear staining after I permeabilize cells with Triton X-100, so it doesn't exist when the cell is still alive or after I fix it with PFA .

Hi, Xiaoman. Unfortunately, I cannot use live cells and I have to permeabilize membranes, so my Ab can enter the cell. Maybe we should try to permeabilize cells with less Triton or another permeabilizer.

Thanks for now.

Best

Stefano

You may try this. It is found in the datasheet.

Have You tried ice-cold acetone? If so, was nuclear staining still there?

Thanks again.

Best

Stefano

Dear Dr. Stefano, how do you stain nuclei? If you stained nuclei with DAPI or Hoechst, during simultaneous scanning fuorescent signal from DAPI may simulate signal from mitotracker. To exclude this artifact you can by using independent scanning of fluorescent signals from each stain.

With best wishes, Nikolay

Dear Dr. Sudakov,

I thanks for your reply. I checked signal bleeding and no DAPI-related signal was leaking into the green channel.

Thanks anyway.

Best

Stefano