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Nuclear Antigen Staining

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safa2410

Participant

Posts: 1

Joined: Mon Feb 20, 2017 7:44 pm

Post Mon Feb 27, 2017 4:06 pm

Nuclear Antigen Staining

Fluidigm has two different staining kits one for intracellular proteins and one for nuclear antigens.... In the nuclear antigen kit they have written that it diminishes the signal of surface staining. I only has 3 nuclear antigens in my panel of 30. I really do not want my surface antigens to diminish in signal on account of these 3 nuclear antigens... Will just the intracellular staining kit be sufficient for these nuclear antigens? Or can you suggest a protocol in which I could successfully stain all three without compromising signal? Thank you.
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mleipold

Guru

Posts: 5796

Joined: Fri Nov 01, 2013 5:30 pm

Location: Stanford HIMC, CA, USA

Post Tue Feb 28, 2017 8:30 pm

Re: Nuclear Antigen Staining

Hi Safa,

Unfortunately, the only real way to find out is to test it.

0) Intracellular cytokines like IFNg and IL-4 are usually fine with saponin, which generally doesn't affect surface epitopes.
1) Even some transcription factors can be seen OK with saponin (I think pS6 was one of them; hopefully Bill O'Gorman and others will chime in)
2) Some transcription factors like the STATs require methanol. However, even post-fixation, I've seen some decrease in surface markers with methanol.
3) Some transcription factors like Foxp3 need even more specialized perm agents. It's been *those* agents that have had the greatest detrimental effect, in my experience.

In short: it depends on your workflow, and your targets of interest. By all means, look at the literature and ask around, but at the end of the day, you just have to see what it looks like in *your* hands.


Mike
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GregBehbehani

Master

Posts: 85

Joined: Tue Apr 12, 2016 10:17 pm

Location: The Ohio State University, Columbus, Ohio

Post Tue Feb 28, 2017 10:14 pm

Re: Nuclear Antigen Staining

Hi Safa,

I would second everything Mike said. I would imagine one of the Fluidigm kits has saponin in it and one has methanol. The methanol will disrupt a large proportion of surface epitopes, but it is better for certain intracellular markers; the STATs being the best known example.

I routinely do a two step stain where I stain the surface markers before premeabilization, then I permeabilize with methanol and stain the intracellular antigens. This works fine in my hands. Fortunately, mass cytometry antibodies are stable in methanol, so the methanol permeabiliaztion won't remove them. I have seen the methanol perm remove some antigens (with the antibody attached), however, so you need to be careful about this. (I assume these surface proteins are just loosely attached to the other membrane proteins and get stripped away when the methanol removes the lipid bilayer.)

Unless a particular STAT is very important for your experiment, I would suggest trying everything with a saponin perm, and if this doesn't give good staining of one or more of your intracellular markers, then go to a two-step staining protocol.

Best of luck,

Greg
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tomash

Contributor

Posts: 25

Joined: Sun Oct 19, 2014 10:15 pm

Post Wed Mar 01, 2017 4:29 am

Re: Nuclear Antigen Staining

Hi Safa,

I wouldn't worry too much about the reduction in signal of your surface markers. Some markers will reduce (although some won't), but for the most part it won't ruin your experiment if you use a perm. The nuclear perms tend to affect surface markers more than the cytoplasmic perms do, but the difference isn't huge. If you can get away with using a saponin-based perm (the one used to perm for cytoplasmic staining/cytokines) then do so, but as the others have said it depends on your intracellular targets: if you are looking at FoxP3, don't bother with anything except a validated nuclear perm. You'll have to validate this yourself, as the worst part is that using different perm methods might free up different amounts of DNA-bound transcription factors (this seems to be the case for FoxP3, for example).

Having said this, things like chemokine receptors or other low expression markers (e.g. CD34 in mouse) will the most heavily impacted by permeabilisation. So as Mike has suggested, if these kinds of markers are the central point of the experiment, then it would be worth considering dropping the intracellular component.

Most protocols/buffers provided commercially involve a one-step fix/perm (i.e. the fixation and permeabilisation occurs simultaneously) that is performed after surface staining and washing. Based on our experience, I would strongly advise you to fix first (e.g. 4% PFA 10 min), then perform the fix/perm (same day or next day) -- in my experience the cells (and the surface marker staining) fare far better if they are subject to the perm AFTER having already been fully fixed. This is something you'll need to test yourself (as I make this recommendation mostly from experience, and only few direct comparisons) but this is what we have found for fluorescence and CyTOF. Note: it is important to still use the fix/perm buffer here for the permeabilisation, as it will contain the special 'nuclear' ingredient (such as Triton X-100) that won't usually be present in the 'perm' buffer.
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jcvillasboas

Contributor

Posts: 41

Joined: Fri Apr 03, 2015 3:22 pm

Location: Rochester - MN

Post Thu Mar 02, 2017 3:43 pm

Re: Nuclear Antigen Staining

Great discussion. I am going to tag along on Safa's thread and ask for specific input for FOXP3 staining on CyTOF. Would you guys mind answering the following questions for me:

1. Has anyone used the eBioscience Foxp3 / Transcription Factor Staining Buffer Set (Catalog Number: 00-5523)?
2. Do you dilute the Permeabilization Buffer (10X) in MaxPar water or just distilled water?
3. How long to you incubate cells with the eBioscience Fix/Perm buffer (DVS protocol suggests 30-45min) before staining?
4. How long to you incubate cells with the nuclear target antibody cocktail (DVS protocol suggests 45-60min)?
5. For those who do a two step staining (surface->perm->nuclear), do you fix your surface targets before permeabilization or does the fix/perm buffer already take care of that?
6. If you are staining for any cytoplasmic targets, can you add the nuclear targets and cytoplasmic targets to the same antibody cocktail and just use methanol-based permeabilization for both?

I did not have luck with the eBioscience Foxp3 / Transcription Factor Staining Buffer Set. My pellet vanished after perm. I wonder if I just incubated it for too long. Any thoughts are appreciated.

Regards

JC

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