What stain do you use for your protein, because it might be either a quenching thing or a problem of gating and overlap between dye emission wavelengths. If you stain a population of cells with just DAPI do you see any "A-Nuclear" cells?

You may need to consider that the cells are simply dead. A couple of years ago when I 1st started using a tissue digestion process and cultured my cells overnight, I never managed to get their nuclei to stain only to find out later that they don't survive for more than a certain number of hours and it wasn't a staining problem but rather a cell viability problem. Hope this is useful.

Try Hoechst 33342, it is better then DAPI. And it can be used for living cell staining.

What stain do you use for your protein, because it might be either a quenching thing or a problem of gating and overlap between dye emission wavelengths. If you stain a population of cells with just DAPI do you see any "A-Nuclear" cells?

I suspect it is the problem of your DAPI. I recommend use more.

Hi.. I thought i might be interesting.. did u repeat it?? and one more thing are all the cells which are not stained with DAPI contain your protein in the nucleus..Also in other word I will also suggest to change the stain use Hoechst 33342 which is better thn DAPI.

HI everyone and thanks for the responses. I've attached an image of the cells in question.

They are primary human vascular smooth muscle cells (Coronary artery). I have the cell conditions down pretty well as far as standard culture and rates of death are pretty low. no evidence of anuclear cells, these cells certainly have a nucleus just not DAPI stained. As I say to my eyes the cell looks like it is processing just fine all with the exception of the DAPI. But it could definitely be that the cell is dead or dying - That is really what I was thinking it is as the Leptomycin B would be ultimately toxic in its nature, the time that this occurs I've no idea of, I guess I will have to do the experiment. It was just a weird observation. and I was wondering if there were any other reasons a nuclei might not stain for DAPI.

I think the most prudent would be to check the cell death first then possibly switch nuclear stain, I've used most of the other stains as well in the past but stuck with this particular DAPI as I got less time dependant bleed through to other channels as you can get with some of the others.

Thanks again for the advice,

S

Also I had consider whether the antibody detection was inhibiting the DAPi but I guessed that might be unlikely as there is absolutely no DAPI - I assume its small enough that it would permeate at least a little

You can use something else like DRAQ-5. But this is strange. Generally, DAPI stains pretty well. There is a combination of mounting media plus DAPI available from invitrogen. This works really well. You can try this if you want.

The picture shows DAPI works. but it seems the cells are not at the same focus level. have you used Z-stack to take the picture? if not, I think that might be caused by light focus.

Scott- this is weird, as you say. Even if your cells were dead, I'd expect positive DAPI staining. My guess is that somehow the fluorophore on your secondary antibody is quenching the DAPI or causing it to photobleach. Have you tried imaging the DAPI channel first?

yes it is the DAPI from invitrogen in mounting gel, it is a Z-stacked image, It is Alexa 594 as the secondary so I'm unsure about the quenching/ bleaching the DAPI here is quite photo-stable. I noted quite a few cells like this under the eyepiece, as I thought it could have been a processing issue and its not a processing issue. all of the ones that had strong nuclear stainig (red) had no DAPI so I guess competition could be an issue. I had heard that a loss of heterochromatin could affect this, but I am assuming it leads to weaker but not no staining. So competition could be an issue. As you say daniel, even when the nuclei "burst" you can usually see fragments of DNA staining, so cell death may not explain it.

found the previous thread below, some other possible explanations, I use prolong antifade gold and it is possible that competition is an issue with the ones stained heavily for the protein:

https://www.researchgate.net/post/Why_in_immunofluorescence_strongly_immunoreactive_nuclei_do_not_stain_with_DAPI1

Hi Scott, actually I was thinking that it is might be result of competetion, your protein when it enters the nucleus it might bind to the place where DAPI stains....so I would suggest you to stain the nucleus with DAPI then do immunostaining with your antibody.

Are you using the correct concentration/fluorescence conditions, like check with filters which you are used? May be problem with medium which may cause increase auto fluorescence (the background).

The fluorophores can still lose fluorescence over time and light exposure

Scott- it sounds like it is worth trying the DAPI stain prior to adding mounting medium. Good catch on the prior thread. Reassuring in some sense that this problem is not unique to your system.

Thanks everyone for the answers, seems like trying different nuclear stains and checking for competition - also possibly dialling down the secondary antibody as recommended by another colleague, might help resolve the issue.

S.

It must be a side-population cell (although its not live staining) or stem cell, may have different DNA confirmation to prevent DAPI binding. I dont think DAPI gets quenched by other fluorphores. I dont think antifade selectively bleach one cell DAPI but not the other. When you do immunocytochemistry, the nuclear export has no chance to operate..so using leptomycin-B cant give answers. I would recommend to proceed with stem cell assays such as immunofluorescence with DAPI vs Oct4 or DAPI vs Sox-2 or DAPI vs Nanog or DAPI vs Alcian blue depending on what you have access to.

OR

Hoechst33342 with your nuclear localized protein antibody to check side-population by FACS with and without Verapamil to see whether your protein high cells match with side-populations

OR

Treat your cells with or without Verapamil and then do immunofuluorescence for your protein with DAPI staining. The Verapamil treated cells should have DAPI in "all cells" but verapamil untreated cells should have some cells without DAPI (similiar to the attached picture in your response).

Hi Goodwin,

All good advice and you may be right that there is a subpopulation of cells within these. They are a bought in primary cell but it is probably not likely 100% contamination free.

Stem cells, maybe, although controversial (isolated from the medial layers of human coronary artery) but still possible. As such its maybe not the most advisable course of action to invest a lot of time in identifying stem cell populations if it is not the focus of these studies- although identifying that it is another population of cells is important. However they do stain positive for SM22alpha actin, which likely makes them smooth muscle cells.

I'm reasonably sure, given that they are cultured cells, that they can be in different states as they have great plasticity as a cell type.

Leptomycin B treatments are done prior to fixation.

Not sure why you are suggesting verapamil, what is it that you are hoping to show with it?

I don't think quenching but I do believe competition for sites could be an issue

Thanks again,

S

Scott- I think Goodwin is referring to adding DAPI to live cells. Stem cells/side populations will exclude the dye. Not true if DAPI is added post-fixation, as in your case.

DAPI cannot be used in live cells because it is not cell permeable. The alternate dye for live cells is Hoechst-33342 (Bisbenzimide) which is cell permeable. In live cells some cells are known to have low uptake of Hoechst33342 and are called side-population cells. Verapamil prevents this so that Hoechst33342 is able to bind DNA in the presence of verapamil.

gotcha and thanks, no the cells I am looking at are all fixed - I appreciate you mean looking in a population to see for stem cell versus not without killing the cells.

Thanks for the advice again

S

Hi Scott,

I use DAPI only as a control staining in my immunhisto- or cytochemical stainings. But I recognized quite often that not all cells are stained by DAPI. I stain mostly for cell membrane proteins which shouldn´t interfere with the nuclear stain. But I do the DAPI staining with the washing after the secondary antibody incubation, so i can´t exclude the competition.

If it is possibly I would try the hoechst (or maybe propidium iodide) staining.

Best luck,

claus

Hi Claus,

Thanks for the info, yes I also add the dapi after 30 mins wash. I may have the same issue as you, it just has never been a problem before. My protein is a membrane protein that I believe translocates to the nucleus to promote cell proliferation. so the important thing really is that the cells with nuclear staining are no dying. That is the crux of my question. Its weird that the nuclei do not have dapi and I can try others butI think the main thing is I can show that it is true staining and not artefact. I will be adding a whole host of other techniques to back it up so time will tell.

Thanks for the help

S

Hi,

would it be an alternative to use an antibody against e.g. a histone or another protein, which is for sure in the nucleus or the nuclear membrane?

Best luck,

Claus

Scott- depending on whether your protein stays resident in the nuclear membrane/envelope throughout mitosis or only in one phase of the cell cycle, you might consider looking at nuclear cell cycle markers as a co-stain. I would recommend Ki67, PCNA, or EdU incorporation (Invitrogen/Mol Probes Click-it proliferation stain).

Hi Daniel,

I'm relatively sure it is G0-G1-S phase transition dependant. I've done quite a few studies in vitro and in vivo using the EDU which is very effective and am enhancing these studies. The question really is, does it enter the nucleus, the answer seems to be yes but I need to find the appropriate ways of effectively demonstrating that. The Leptomycin should remove the effects of fast shuttling of proteins in and out of the nucleus (at least that is the hope) I am backing up with a few other techniques as well as treatments to stop intracellular transport (from the membrane) as well.

Thanks again for the input,

S

Scott- some other approaches that could help out your study are to try nuclear fractionation to examine distribution of your protein by Western blot, and to track your protein in live cells using HaloTag technology (rather than indirect immunofluorescence). Good luck!

thanks again Daniel,

I've done the Westerns on fractions which work fine. I'll need to take a look at HaloTag, thanks for the tip

S

Hi,

i have the same observations, i use DAPI as a nuclear staining in my immunhistochemical stainings (by using DAP +CHROMOGEN FROM dako). The cells with nuclear staining of my protein are no dying. i used several cell lines and i had the same observations....

Scott, did you understand the mechanism or did you resolve your problem??

tkS

Hi Fadi,

I have not had the chance to fully work this through at the moment but as others suggested I switched to a different nuclear marker (Hoescht), the DAPI I was using was fairly mild and low intensity stain. The hoescht is a lot brighter. I should point out that it wasn't just my protein of interest antibody that worked in this way, there were a number of other known nuclear locating proteins that I found with this. So I believe its likely some kind of detection issue rather than cell death issue.

Like you said, my evidence points to my cells not dying, but I have not fully followed up this aspect at this point. I've been working on some different ways to mark this (as opposed to antibody detection).

Best,

Scott

Hi

What is the seconday antibody that you have used to stain your protein? Just to be sure that things are alright (I don't know, you might have already tried), you can try two things, change the colour of the secondary antibody or try the same secondary antibody with another common sure shot nuclear protein like histone before staining with DAPI and see if you still have the same problem.

Best, Suparna

When I use DAPI on tissue sections, 5 min of incubation yields bright staining. However, it takes much longer to stain cultured cell nuclei.

Maybe, you just need to incubate longer with DAPI solution.

Hi Hong-Xiang,

I agree DAPI can create bright nuclear staining, it depends on the stain that you are using, if it is DAPI diluted at varying concentrations in PBS it can be very bright (and can also cause issues with bleed through in other channels). I am using the DAPI solution from Invitrogen (prolong anti-fade gold) which stains nuclei well, but is fairly dilute which as you say can be bad for very bright staining but good in that it doesn't bleed through the other channels.

Thanks for the suggestion.

Best,

Scott

Thanks, Scott!

I do not use DAPI prolong gold anti-fade. Actually Invitrogen technical people suggested us not to use it at a workshop.

Incubation with DAPI solution in PBS gives us flexibilities. We can easily control the concentration and incubation time for an optimal staining of the nuclei. I have never seen the bleed-through of DAPI to other channels.

It's worth a try and good luck for your alternative ways for nucleus staining.

Hongxiang

Hi Scott,

I am facing the same problem in my imaging experiment wherein I see very faint (or none at all) DAPI staining in my GFP transfected cells, treated with LMB; however, the DAPI staining is quite prominent in non-LMB treated cells. 

Were you able to fix your problem? Please let me know the way around this problem, if you found any.

Thanks,