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How to maintain cells at a specific apoptosis stage - (Mar/07/2009 )

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With DAPI and fluorescence microscopy you can fix beforehand and then permeabilise the cells with a 10 min incubation in PBS or TBS containing 0.1% Tween 20 or Triton X-100 to allow the dye to enter the cells.


NemomeN on Mar 9 2009, 10:11 AM said:

Stain your cells with your Annexin antibody and wash appropriately and then fix them in formaldehyde (anywhere from 1 to 4%). You can keep them at 4 degrees until your run. You may want to play with the formaledhyde concentration to keep the strength of your signal. If you fix before you stain, you will get massive false positive. What i am not certain about is the counterstain (ie. propidium or 7-AAD), but at least you can keep your annexin V signal.

If I were you, I would try this method. But I am concerned about dying out of fluorecence during the treatment. Since in some flow cytometry analysis, intracellular antibody were used (Oct4 as I know). And I remeber the cells need to be fixed. And you could count on some related litterature to optimal your condtion. May it help you and good luck.


DAPI will work, but you will need to permeablize after you fix so that it can enter. If you fix before you stain, your annexin signal will be all false positive

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