I need to gate eosinophil on FACS by using double markers CDw125 (surface markers) and EPX (intracellular proteins). Here I already had PE antiCDw125, but for EPX, I need to do indirect staining. Is it possible for me to perform dual staining in one sample?
Firstly, I intend to stain surface marker (PE antiCDw125), then fixation and permeabilization. Then, I would incubate cells gradually with primary and FITC-secondary antibody for EPX. Does it sound feasible? And how can we prevent non-specific bindings from secondary antibody?
Could you please give me your opinion? Thank you all.
Hi Tu Trinh,
as Holger mentioned already that should not be a problem! First stain the outside, then fix and perm. Fc Block is great for your surface staining, for the intracellular staining its not so important. Here I would prefer to block the cells with serum to prevent unspecific binding.
Although I think it is clear, just to be sure one more thing: Take care that the secondary antibody you are using for your intracellular staining is not binding also to your CDw125 mAb. For example if the CDw125 is rat anti-human (or mouse) then your secondary mAb should not react with rat mAbs.
One last thing: make sure that you have the proper controls. For the ic staining you'll need at least one sample were you use an isotype control or just stain the secondary mAb.
Hi
As others mentioned first stain the cells for surface marker and then fix the cells with 4% para formaldehyde and the perm it with 0.2% saponin with sodium azide to block the metabolism. Use Fc block to prevent non specific binding and use secondary antibody as your isotypic control.
Blocking Fc is very important in preventing the non-specific binding. You can use either the commercial Purified Human FcR-Inhibitors or 1% mouse serum
It does sound feasible and sensible an approach.
People get weary reagrding indirect staining for intracellullar proteins, as well as regarding intracellular staining with polyclonals. We just detected (specifically) an intracellular protein with a polyclonal. Fc blocking, as Moses mentions, is essential. Alos, you can use EPX-negative cells as specificity control (they shouldn´t bind your antibody.
AND if there are EPX specific monoclonals available, which are not conjugated with a fluorochrome, you can try labeling them yourself. Innova Biosciences is apparently good at that.
Hope this info serves.
Thank you all for your suggestion!
Since Fc blocking is really necessary, I should definitely apply it.
In addition, I tried to fix cells with 2% PFA for 10 min at room temperature and then permeabilize with 0.2% saponin with 5% FBS for 10 min, room temperature. However, cell morphology changed a lot after those steps. Would those changes affects quality of staining? As a results, would it affect FACS results?
Here I enclosed 2 pictures of my results for your reference. I appreciate any of your suggestions.
According to my experience, the morpophological change is unavoidable. Saponin disrupts lipids in the membrane, exposing internal structures of the cell, so "permeabilization" is not just permeabilization.
Hi, the simplest identification eosinophils in blood cells is using surface CD45 antibody - dotplot SSC-A vs CD45-A. I enclosed picture.
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