I work with human mo-DCs and I need to detect cytoplasmatic and nuclear proteins by flow cytometry using flurochrome-conjugated antibodies (direct detection), that is, I need to permeabilize both plasmatic membrane and nuclear membrane. Is it possible to use the same permeabilizing agent to perform intracellular staining of cytoplasmatic and nuclear proteins in flow cytometry ? Or necessarily I need to perform different procedures to these two kind of proteins?
If it is possible, someone can provide a protocol or suggest a permeabilizing agent to this aim?
Hi,
You can but it mostly works with cytokines and transcriptions factors combination. Since plasma membrane would be compromised you should use appropriate antigens as well as fluorochrome combination which will not affect the loss due to plasma membrane damage. I have also done with cell signaling proteins. BD and ebiosciences has variable buffers you can try those.
We immunostain cytosolic and nuclear proteins all the time. Formaldehyde fixation then Triton x-100.
Matt Jackson Can I use then Triton X-100 to detect nuclear and cytoplasmatic proteins without loss of soluble cytiplasmatic proteins?
As long as you fix it with formaldehyde first. You want the stuff with methanol stabilizer to porate it and get the formaldehyde inside the cell. Shouldn't be an issue. The nonstabilized stuff is a speciality item.
I personally have tried commercial solutions that are for the one or the other purpose, but it didn't work.
I haven't tried the solution that Matt Jackson provides though. In that case you don't need to buy any commercial kits at all. I have tried this combination only for tissue sections (from OCT blocks) and works nicely there for various proteins I needed to stain.
Otherwise, the eBioscience Foxp3 kit works nicely for intranuclear staining (transcription factors etc), while the BD Cytofix/Perm kit works nicely for intracellular staining (cytoplasmic proteins or secreted proteins blocked with Monensin or Brefeldin A).
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology
- Antibodies