What's the best type of fluorophore to use for targets that are in the nucleus of the cell?
To test this, I'm using human PBMCs and staining for T-bet, which is a transcription factor. I've done this with overnight staining at 4C, using BD's Cytofix/Cytoperm kit. This kit isn't designed to work for the nucleus and in short staining times, it doesn't. This is going to allow us to see a neat aspect of intracellular staining, which is the relationship between the type of fluorophore used and the quality of staining obtained.
Let's start with the classics: PE-based fluorophores to give us maximum brightness.
Human PBMCs stained overnight after fixation with BD Cytofix/Cytoperm. Top row: FMOs. Bottom row: staining with anti-T-bet antibodies. Clone indicated in parentheses.
We get a bit of background binding with PE-tandem conjugates sometimes. This can usually be fixed by optimizing the dilution factor with titrations. For this test, I'm using all the antibodies at the same concentration, except for the REA102 because I don't know the concentration. The REA antibodies are also engineered to have reduced non-specific binding, and this can help quite a bit.
How about Alexa Fluors and others like this?
I'm under the impression that eFluor 660 and R718 are both relatives of the Alexa Fluors, but I could be wrong. KIRAVIA Blue 520 is quite different--it's a much larger long-chain dye. I'm including it here because it's in a class of its own and I need to flesh out the figure.
These are all working, but the background is much higher with the AF594. eFluor 660 is quite good at giving us separation of all the different levels of T-bet (could probably be better with a bit less antibody). R718 probably isn't quite as bright, but the background is minimal. That's good.
How about the Brilliant dyes? They're really bright, right?
These don't work well for nuclear epitopes, from what I've seen. Why? Well, they're quite big, so they may not fit through the small holes to get into the nucleus. There's not much info available online (or from BD), but what we can glean from BioLegend's website suggests that conjugating an antibody to a Brilliant Violet would add about 290kDa to its mass, tripling the size of the antibody. PE, though, is about 240kDa, and that works fine. So, it isn't just about size. Unlike PE, Brilliant dyes are (probably?) long-chain polymers so my guess is that they get tangled up like bits of yarn. PE and particularly its tandems do have some issues with background for nuclear staining, though, which is consistent with size being a problem. If they're too big, they may get in eventually in an overnight incubation, but may have trouble getting out again in a short wash protocol.
You can use Brilliant dyes with other protocols that more effectively permeabilize the nucleus, but they aren't going to be your best choice even then.
Where big is bad, small is king. The Real dyes from BD are small molecules as well, apparently.
Real Blue 545 is the dimmest option. I would say the separation is as good as PE. The Real Blue 705 among the brightest (Real Blue 780 and Real Blue 613 are probably brighter on the Aurora). With both, there's not much background, suggesting the antibody can both get in to find the target and that any excess can be effectively removed with normal washing steps.
So, to sum up, if you're having trouble staining a nuclear antigen, try a conjugate with a bright small fluorophore. A nanobody would probably also be a good bet.
Antibodies in this post:
Resplendent Quetzal, Costa Rica