From:Tom Clarke

I've had some pretty poor luck with GFP and paraffin sectioning, so be warned
that you might lose most of the GFP expression. The difficult part is
apparently the ethanol steps - the strong denaturing of protein that this
causes either damages the GFP protein irreversably or washes it out of the
cell.  The type of GFP protein that you use seems to make a difference -
enhanced GFP (eGFP) is supposed to work better then GFP and membrane anchored
eGFP is thought to be more stable then eGFP that stays in the cytoplasm.

In my own experience using eGFP expression to locate viral infected cells,
most of the fluorescence is lost, but if the cell was strongly GFP positive,
some residual fluorescence remains.  Weakly fluorescent cells lose all
detectable GFP expression after paraffin processing, however.


Tamara Howard wrote:

> Jenny -
> GFP (Green Fluorescent Protein) may make it through paraffin processing
> and still fluoresce - it seems to depend on the construct itself - so I'd
> give it a whirl and see if you can still get glowing whatever it is that
> is expressing. If your construct does not like being dehydrated and
> heated, there are several antibodies to GFP available, but the same caveat
> seems to apply - depends on the construct. Some people have had great
> success with this; others have headaches & nightmares :)
> The bottom line is, does your researcher really need paraffin sections? No
> access to a nice confocal or multi-photon system on which you could do
> whole mount or vibratome slices? Life would be much easier!
> Good luck.
> Tamara
> |--------------------------------------------------|
>  Tamara Howard
>  Department of Cell Biology and Physiology
>  University of New Mexico - Health Sciences Center
>  Albuquerque, NM 87131
> |--------------------------------------------------|

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