Followup: Re: Good fluors for intracellular

Thank you for the several responses.  I get the impression that most 
molecules will work, provided you are slightly more aggressive with 
permeabilization.

I s'pose it remains as we often say, each experiment must be judged on its 
own merits.

-cb



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The conventional wisdom for intracellular staining held that anything 
except FITC was "too big" to get inside the cell. With improved methods, 
this is no longer the case. Caltag's Fix & Perm works beautifully with PE 
and APC, and in fact we recommend PE conjugates whenever possible because 
you're usually looking for a "needle in a haystack" and using the brightest 
fluorochrome improves your resolution. The only fluors that don't work well 
are the tandem conjugates, because with them size does become an issue. You 
can use tandems, but it's much harder and I don't advise using them unless 
you really can't use anything else. The direct-labeled Alexa Fluor 
conjugates work very nicely with Fix & Perm as well. I just got some data 
from one of my customers showing beautiful staining for IL-4 Alexa Fluor 488.
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You should increase your saponin concentration to 0.1% to 0.5% for good
permeabilization. Although the bd cytofix/cytoperm buffer works well for
intracellular stain, the staining benefits from a second perm (bd perm
wash) and used as the buffer for the staining cocktail.
With triton, 0.1% would be good.


PE and APC get into the cell quite well. PE is beneficial, although
larger because it is the brightest fluorophore.

Alternatively the alexa dyes are excellent for intracellular stain, and
anti-cytokine alexa abs are already on the market.
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We use coulter perm reagents (formaldahyde-based) with our clinical
samples. All flourochroms we have work well (FITC, PE, PC5, ECD).  The
only drawback is cells loose FS signal (fixed/dead).  CD45/SS gating is
a must.
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PE and APC work fine. The question is whether biotin followed by SA-PE 
works. This seems to be suboptimal due to size but may work if you incubate 
for a long time. We have found that biotin followed by SA-Alexa647 does work.
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