From: Maryalice Stetler-Stevenson (stetler@box-s.nih.gov)
Date: Thu Jul 30 1998 - 08:49:26 EST
I got several replies from others who have tried to fix Annexin V stained cells. One was positive and the others negative. I have cut and pasted the replies below. 1. I have used annexin with fixation with reasonable results, see Manion and Frey, Cytometry (CCC) 26:317-322 fig 3 for data. The combination of no wash and fixation did degrade staining in that figure. The key trick is to supplement your fixative with divalent cations, we use FACSlyse with 1 mM calcium. This has worked in several studies for me. A second key observation is that annexin binding is reversible even after PFA fixation, any washes also need to be done in calcium supplemented buffers. 2. Hello,We have tried fixing cells after annexin V staining, in PBLs. Our experience is that the level of apoptosis that we see by annexin is less after fixation than if we analyse cells that are freshly stained. Therefore, if you need to use a fixation step, I suggest that you think of using a tunel stazin, which in our hands is quite amenable to fixation. 3. We've not convinced ourselves that any fixation technique (particularly EtOH) is compatible with Annexin-V staining. All pre-fixes have been a bust, and post-fixing does not give us the same results. Our experience, however, is limited to tissue culture cell lines- mostly ones growing attached to plastic. 4. >I tried fixing stained cells following the protocol from Techniques in Apoptosis: A User's Guide: T. Cotter and SJ Martin 1997, page 113, but without good results. As the authors say in that book, fixation procedures will allow any free annexin v to enter the cell and bind to PS on the inner leaflet of plasma membrane. From my own experience, the best way to do it is to have a small number of samples and to analyse them as fast as you can. Keeping them on ice alters the results as well. >For detection by immunofluorescence microscopy in tissues, once you >section the tissue, you will expose PS from the inner leaflet and get >false positives. > Maryalice Stetler-Stevenson Director Flow Cytometry Unit Laboratory of Pathology, NCI, NIH
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