I did an immunofluorescent experiment with both unstimulated and stimulated MC/9 cells and stained for CD107a. I didn't permeabilise the cells, however, I noticed strong intracellular staining in unstimulated cells (CD107a is inside the cells, so I shouldn't have seen any staining). Does anyone know what happened?
Hello Diliana Stanolova,
One possible reason that comes to my mind is the type of fixative that you have used. Organic solvents such as methanol and acetone are used as fixative since they can coagulate proteins. But they can also dissolve lipids from cell membrane making them permeable to antibodies.
So, cells undergoing acetone or methanol fixation does not require a permeabilization step. Therefore, even if you haven't permeabilize the cells, by using such fixatives, you have unknowingly permeabilized the cells.
For immunofluorescence, fixation with 4% formaldehyde is recommended.
Best.
Why did the antibody cross the cell membrane?
I'm tempted to say 'to get to the other side'
But I shouldn't joke. Sorry.
Malcolm Nobre Thank you for your answer. I used 4% PFA to fix the cells, so it can’t be because of the fixation.
Damage from exposure to air during fixation, rinsing and antibody incubation can permeabilize the plasma membrane. Especially a problem working in a laminar flow hood. Cover the dish between steps. Also old paraformaldehyde solution makes formic acid, which can damage the membranes. Citrate antigen retrieval can also do this.
Hi Diliana,
Some researchers suggest using of fixation only for staining nuclear proteins. So, antibodies can enter the cell even with no fixation.
But I am wondering what type of imaging did you use (confocal or fluorescence microscope)?
Mohamed Khashan I did both. And I noticed the same situation with either.
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