Annexin V staining in combination with other antibodies
Posted 24 August 2009 - 12:26 AM
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?
Posted 24 August 2009 - 03:47 AM
Posted 24 August 2009 - 04:33 AM
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?
Posted 24 August 2009 - 03:50 PM
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?
Posted 24 August 2009 - 11:32 PM