4 replies to this topic

[James Green]

Hi,

I want to stain some cells for annexin v and then stain for a protein of interest (nuclear). Is this a case of simply fixing AFTER annexin v staining and then coming in with my primary antibody? Does the fixing process degrade annexin v and should I use something other than 4% PFM?

Regards,
James

[Stephan]

It would depend on what each of your antibodies are labeled with.  I would suggest two very different fluorochromes to avoid confusion but I'm not so sure how much the antibodies would degrade with fixing.  They are quite fragile so im sure they wouldn't persist after fixing.  Someone here is using 7 different antibodies simultaneously to sort out specific populations so multiple antibodies are definitely possible, just not sure exactly what you are interested in doing.

[James Green]

Thanks,

I want to stain with annexin v after expressing an apoptotic protein and then resue the phenotype with expression of another protein.

Thus, I would like to stain for annexin v on the cell surface (green), fix cells (4% PFM), permeablize cells (with 0.1% triton x-100) and then stain for the other two proteins of interest (blue and red). To view by confocal.

I'm just not sure if the fixing process or the permeablization will effect the initial annexin v staining?

[miBunny]

it sounds dicey... remember that annexin V recognizes a phospholipid normally on the inner leaflet of the plasma membrane.  Early apoptotic events result in a flipping of that lipid to the outer surface of the plasma membrane where it can be bound by annexin V.
Permeabilizing the cell will open up the membrane and that normally hidden phospholipid (can't remember which one) will be available for binding by annexin V.  It is possible that all cells could come up annexin v positive.

If you are going to be permeabiizing the cells, why not just stain for an intracellular marker of apoptosis such as cleavaed caspase 3?

[James Green]

Thanks for the help. I think staining for active caspase 3 is a much safer bet.

Thanks again,
James