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Immunofluorescence using dead cells?


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#1 Tn1

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Posted 21 September 2010 - 01:40 AM

Hi everyone,

I'm searching for a possibility to stain dead cells (from the oral mucosa). I DON'T want to distinguish between dead and live cells, as I already know all of my cells are dead. But I would love to be able to perform a normal immunofluorescence ON these dead cells. We tried this once (with antibodies staining the golgi and tubulin) but no signal could be detected.
So my question is: IS there a way to perform immunofluorescence with dead cells? And if there is: How can it be done?
As we don't really care about WHAT we stain within the cell, (we just want to stain anything- and if possible visualize at least three different color signals. Maybe an organelle and the cytoskeleton would be great ;-)) any possibility would be very helpful!! (We already know DAPI works with dead cells but besides we have no clue).

It would be great if anyone could help us!

Thx and greetings from Germany!

#2 Piersgb

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Posted 21 September 2010 - 07:35 AM

I guess the problem with immunostaining of dead cells is that upon the initiation of apoptosis and/or necrosis the cells may undergo a lot of surface and intracellular membraneous protein changes. In other words, as soon as the cells start dying they may begin to shed the normal antigens that you would expect to get in the usual places.

So I guess you could try and catch these cells to as near as death as you can and then fix them with something like 4% Paraformaldehyde (PFA) in 1xPBS. This would preserve surface antigens. If you wanted to get to the intracellular stuff you could permeabilise them with a detergent such as Triton-X100. Not sure if this would help the PFA get in and fix the interior of the cell.

The other option I guess would be to try and find some antigens that continue to exist and thus allow you to stain after the cells die.

Just my two cents!




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