I want to do some co-localization analysis between fluorescent-protein-fused gene and the antibody of the gene, to prove the correct location of the fluorescent-protein-fused tool during live cell imaging.
However, I found that the transfection signals just lost after IF assay. And I detected the signals step by step during IF, which told me that permeabilization would greatly kill the transfection signals.
I usually use 0.1% Triton treat the cells for 3min.
Do you have any similar experiences? Can you give me some suggestions?
So many thanks!
A eukaryotic cell is separated from the extracellular environment by a plasma membrane composed of a phospholipid bilayer containing proteins that regulate transit of molecules into and out of the cell. Loss of this barrier function can lead to compromised cellular homeostasis and death of the cell Lu Zhuang
sometimes, there is no need for permeabilization. I have experienced that and it worked perfectly. Some cell lines exhibit big membrane holes, thus easy to penetrate.
Hi there Lu Zhuang
Could you please elaborate on your IF protocol. Normally, after fixation the permeabilization with 0.% Triton should not lead to exhaustion of transfection signal. Could you also specify which fluorescent protein tag you are using here?
We do this routinely, and have not really faced a loss in fluorescence of the tag upon 4% PFA fixation and permeabilization with 0.1% Triton (even if its for an hour).
However, if you are not properly fixing your cells, or performing permeabilization on live cells, the cytosol will flow out (if your protein is cytosolic and in soluble state, it will be lost). Some staining protocols, require cytosol drainage and in those cases the permeabilization is done on live cells.
Also if you are fixing with organic solvents, it will kill the fluorescence of pretty much any fluorescent protein.
Hope this helps
Best
Aparajita
- Badges
- Science topic
- Similar topics
- Correction